2013 Awards Apotex Fermentation Inc

Graphic Design: www.aniko.ca Welcome and congratulations on participating in the 2013 Canadian Student Health Research Forum, a one-of- a-kind venue offering students doing health research the opportunity to network, learn about cutting-edge research from internationally renowned experts and be recognized for their own outstanding scientific accomplishments.

The theme of this year’s symposium Medical Student Research Symposium is 'Big Science, Informatics and Bio- and the CIHR National Research Poster medicine'. In an increasingly complex Presentation. We trust that the valu- world, researchers are relying more and able connections you make with your more on tools for generating, synthe- peers, judges and CIHR leadership sizing and using biological knowledge. will provide career-enhancing oppor- We are proud of our exciting line-up of tunities as well as a fun and enjoyable speakers who will share with us their experience. A big thank-you to all the latest discoveries in applying biological student groups for their energetic sup- imperatives to clinical practice. We’re port in hosting our visitors. Brian Postl, MD confident that your own research pro- Winnipeg is an exciting and vibrant Dean grams will be inspired by the wealth of place to live, work and conduct state - knowledge and experience our speak- of-the-art health research. We hope ers bring to this forum. you enjoy your visit of the University of We extend warm greetings and a Manitoba campus and have the oppor - 'friendly Manitoba' welcome to all par - tunity to explore all that our city has to ticipants from across Canada who are offer. taking part in the Canadian National symposium programme Symposium on Big Science, Informatics and biomedicine

Thursday, June 6, 2013 8:00 registration & Refreshments (Concourse) 9:00 Welcome and Opening Remarks Frederic Gaspard Dr. Brian Postl, Dean, Faculty of Medicine, University of Manitoba Lecture Theatre, 9:10 Chair’s Overview Dr. John Wilkins Basic Medical Sciences 9:15 Dr. Thomas Gingeras – Cold Spring Harbor Laboratory, Laurel Hollow, NY Building, “ Communication Inside and Outside Cells via RNA” 730 William Avenue 10:00 Coffee Break (Mezzanine) 10:30 Dr. Curtis Huttenhower – Harvard School of Public Health, Boston, MA “ Bug Bytes: Bioinformatics for the Human Microbiome in Health and Disease” 11:15 Dr. Leroy Hood – Institute for Systems Biology, Seattle, WA “ A Systems Approach to Disease, the Emergence of Transformational Technologies, the Challenge of Big Data and Proactive P4 Medicine” 12:00 lunch and Poster Session (Brodie Atrium) 12:30 Presentations of Recognition (Brodie Atrium) Graduate Students Assoc. Award for Distinction in Mentorship Karol D. McNeill Research Technician Award Dr. Kenneth Hughes Young Investigator Award Dr. Aubie Angel Young Investigator Award Faculty Recognition for Outstanding Contribution to Research 14:00 Dr. Marco Essig – Heidelberg Medical School and University of Erlangen, Germany “Data and Protocol Management in Functional MR Imaging of the Brain” 14:45 Coffee Break (Mezzanine) 15:15 Dr. Iain Fraser – National Institute of Allergy and Infectious Diseases, Bethesda, MD “ Exploring Immune Signaling Systems with High Throughput, High Content Screening” 16:00 round Table Discussion

16:30 awards Ceremony

17:15 reception (Concourse) 2013 Awards Apotex Fermentation Inc. Award – Molecular Biology Canadian Institutes of Health Research – Poster Presentations Children’s Hospital Foundation Award – Child Health Dean of Graduate Studies – Poster Presentations Dean of Medicine – Poster Presentations E.L. Drewry Memorial Award – Health Research Graduate Students Association – Poster Presentation Hain, Emil and Lynette Scholarship – Excellence in Oncology Research Health Sciences Centre Foundation Inc. Award – Infection & Immunity/Neurobiology Manitoba Health Research Council – Postdoctoral Fellow Poster Presentation Manitoba Medical Service Foundation Inc. – Poster Presentations – Awards for Excellence in Research S.W. Prowse Memorial Award – Clinical Research St. Boniface General Hospital Research Foundation Inc. Award – Cardiovascular Biology The Winnipeg Foundation – Travel Awards 2013 Organizing CIHR/National Manitoba Poster Awards Committee Poster Judging Panel Judging Panel Committees Patrick Choy Co-Chairs, Don Smyth, Micheal Czubryt, Chair Stephanie Chu Frans van der Hoorn Adam Burgener Kevin Coombs Ratna Bose Eftekhar Eftekharpour Major Awards Michael Czubryt Serge Desnoyers Brenda Elias Committee James Davie Janice Dodd Jean-Eric Ghia Yvonne Myal, Chair Serge Desnoyers Vernon Dolinsky James Gilchrist Cindy Ellison Hani El-Gabalawy Jules Doré Joe Gordon Paul Fernyhough Jenifer Ezirim Brent Fedirchuk Andrew Halayko Keith Fowkes Andrew Halayko Dan Gietz Sabine Hombach Elissavet Kardami Grant Hatch Sari Hannila Tammy Ivanco Etienne Leygue Kent HayGlass Tiina Kauppinen Hassan Marzban Heather Innis Sohelia Karimi Jonathan McGavock Robert (Bob) Shiu Edwin Kroeger Sachin Katyal Kirk McManus Barb Triggs-Raine Gerald Minuk Thomas Klonisch Adrienne Meyers Jude Uzonna Yvonne Myal Donald Miller Tooru Mizuno Jeff Wigle Peter Nickerson Grant McClarty Jim Nagy Kimberley Ormiston Jason Peeler Thomas Netticadan Don Smyth Bob Shiu Mojgan Rastegar Terri Turner Louise Simard Julia Rempel Frans van der Hoorn Abdel Soussi Gounni Barb Shay Carolyn Weiss Jude Uzonna Bob Shiu John Wilkins Jun-Feng Wang Miyoung Suh Jeff Wigle Bob Tate guest speakers

Marco Essig Iain Fraser Thomas Gingeras Leroy Hood Curtis Huttenhower Marco Essig is professor of Radiology at Heidelberg Medical School and currently works as a radiologist and neuroradiologist at the University Hospital in Erlangen. He received his medical degree from the University of Heidelberg and his doctorate in neurological sciences at the Medical Faculty of the University of Heidelberg.

He is co-founder of Radiology-Proto‑ ing the Deutsche Rontgengesellschaft, cols, an online protocol database and the International Society for Magnetic protocol management system. Dr. Essig Resonance in Medicine, the Radiological is author on more than 200 peer- Society of North America, the Deutsche reviewed articles and author of more Gesellschaft für Neuroradiologie, the than 20 book chapters. American Society of Neuroradiology, Dr. Essig did his internship and resi- the European Society of Neuroradiol- dency training in radiology at the German ogy, and the European Society of Radi- Cancer Research Center. He pursued two ology. He is member of the editorial Marco Essig, fellowships, one in neuroradiology at the board as well as a reviewer of a number MD, Phd University of Iowa Hospitals and Clinics, of prestigious journals. and a second in interventional radiology In 2008 he was appointed as the University of Heidelberg at Brigham and Women’s Hospital, Har- deputy editor for Neuroradiology of the Medical School, vard Medical School. Journal of Magnetic Resonance Imaging Germany In 2006 he was appointed as full (JMRI). Beside his clinical appointment Professor of Radiology at Heidelberg he works as a consultant for pharmaceu- Medical School. Dr. Essig is a valued tical companies and contract research member of numerous societies, includ- organizations. Data and Protocol additional functional assessments. Management in Functional Functional imaging adopts models from nuclear medicine or other physi- MR Imaging of the Brain ologic measurement techniques to The goals and requirements for CNS noninvasively assess parameters like imaging are making a diagnosis and / or perfusion, permeability of blood ves- a differential diagnosis, while accurate sels, metabolics, flow, tissue integrity lesion grading and delineation is needed and many others. Most techniques in case of e.g. tumor description and risk require the acquisition of images over assessment. Imaging is also involved in a longer period of time or during the the decision-making process for ther- injection of contrast media, resulting apy and later for precise planning of sur - in a large amount of imaging data that gical or radiotherapeutic interventions requires a standardized and quantita- which both requires an optimal detec- tive post-processing. tion and delineation of the lesions. After To use the results of functional imag- therapy neuroimaging techniques have ing in a clinical routine set-up the imme- shown to be mandatory for monitoring diate assessment of the often very large of disease and to identify and monitor datasets is needed and an intuitive data possible therapy related side effects. presentation needs to be developed. In MR imaging initially focused on the this presentation the current available superb contrast and spatial resolution functional imaging techniques will be of neuronal tissue to enable a detailed reviewed and innovative post-process- morphological analysis. Recent devel- ing and data presentation techniques opments, however, have focused on presented. evaluating these in conjunction with Iain Fraser is Chief of the Signaling Systems Unit in the Laboratory of Systems Biology at NIAID, NIH. He received his B.S. in biochemistry from Heriot-Watt University, Edinburgh, and his Ph.D. in biochemistry from Imperial College, University of London.

he was a Wellcome Trust International in a complex environment to ensure postdoctoral fellow at the Vollum Insti- context-dependent cellular responses. tute and co-director of the Alliance for This is vital to understanding how a Cellular Signaling (AfCS) molecular breakdown in information processing biology group at the California Institute through cell-surface receptors and their of Technology. linked signal transduction pathways his research has focused on the leads to human disease. Dr. Fraser has mechanistic basis of cellular signaling, developed sophisticated approaches both in G protein signaling networks for RNAi-based perturbation analysis of and more recently in Toll-like receptor immune cell signaling, as well as cloning signaling in innate immune cells. He is platforms and plasmid repositories for IaIn Fraser, PhD interested in the application of sys- high-throughput imaging and cell bio- National Institute of Allergy tems biology approaches to decipher logical applications. and Infectious Diseases, how mammalian cells integrate stimuli Bethesda, MD Exploring Immune responses to infection or vaccination Signaling Systems with and can be used to design new therapies or vaccines. We use high-throughput High Throughput, High genome-scale technologies (such as Content Screening siRNA screening and profiling of RNA expression and protein modification) Modern technology now allows the to broadly characterize the cellular analysis of immune responses and host- response to pathogenic stimuli. pathogen interactions at a global level, i will describe data from ongoing across scales ranging from intracellular projects in our lab including: signaling networks to individual cell • Genome-wide RNAi screens to char- behavior to the functioning of a tissue, acterize signaling network topology an organ, and the whole organism. The in hematopoietic cells and to identify challenge is not only to collect the large the 'parts list' of cellular components amounts of data such methods permit, involved in immune cell responses. but also to organize the information in a • Development of multicolor cell-based manner that enhances our understand- fluorescence assays for screening ing of how the immune system operates applications and parameterization of or how pathogens affect their hosts. computational models. Our laboratory is part of a systems biol- • Use of the above methods to charac- ogy program at the NIH whose goal is to terize the response of immune cells develop detailed quantitative models to defined stimuli, such as single and that can be used to predict the behavior combined pathogenic ligands, and of a complex biological system, whose intact pathogens, such as bacteria properties help explain the mechanistic and viruses. basis for physiological and pathological Dr. Gingeras received his Ph.D. in Biology from New York University (1976). Subsequently, he received his postdoctoral training at Cold Spring Harbor Laboratory under Nobel Laureate, Dr. Richard Roberts as an American Cancer Society and National Institutes of Health Fellow postdoctoral fellow.

After this postdoctoral training period Laboratories serving as Vice Presi- Dr. Gingeras became a Staff Scientist at dent of Biological Sciences. Currently Cold Spring Harbor Laboratory. He then he is Professor and Head of Functional accepted the position of Senior Scien- Genomics at Cold Spring Harbor Labo- tist at the Salk Institute Biotechnolgy/ ratory and Adjunct Professor at the Industrial Associates and became the Department of Genetics at State Uni- Assistant Laboratory Director and versity of New York at Stony Brook and Director of Molecular Diagnostics at Tsinghua Universities. He is the author SIBIA. In 1990 Dr. Gingeras became and coauthor of 158 manuscripts and Thomas Director of Life Sciences Research Lab- 29 patents. His laboratory is currently Gingeras, phd oratory for Baxter Heathcare, Inc. and focused on the organization and regula- Associate Adjunct Professor, Depart- tion of functional information encoded Cold Spring Harbor Laboratory, ment of Pathology, School of Medicine, in genomes and the biological roles of Laurel Hollow, NY University of California, San Diego. In non-protein coding RNAs. 1993 Dr. Gingeras moved to Affymetrix Communication Inside lessons such as: a) expression ranges and Outside Cells follow transcript types and subcellular localization b) expression of isoforms of via RNA a gene by a cell do not follow a minimal- DNA represents a storage device istic strategy, c) genomic characteris- encoding information for hundreds of tics of potential trans-acting enhancer thousands of processes. This informa- regions are distinguishable from other tion is partially revealed in the RNAs types of cis-acting regulatory regions d) that may be composed of 12 billion expression status and expression levels nucleotides. Results from the human can be predicted by different groups of (ENCODE), fly-worm (modENCODE) and chromatin features with high accuracy mouse ENCODE projects have shed light and e) pervasive genome-wide tran- on both the functional content and how scription prompts a need to redefine the this information is organized. There are definition of a gene . Finally, the infor - a total of 194,034 transcripts annotated mational program encoded in genomes within 56,563 genic regions represent extends outside of the cell boundaries. our previously best manually-curated The portions of the human genome annotation (based on v16 Gencode). selected to be exported outside the cell The results from the ENCODE project in the form of RNAs are highly selective point to considerable supplementation and influential. Thus, selective portions of these data including the location of of genomes are being used as part of an regulatory regions, the association of intercellular communication process human variation with various types and are produced by almost all cells of functional domains and the role of thus far examined. The ENCODE data epigenetic modifications on the func- sets also point to the need for new and tionality of various elements. Analyses robust computational approaches to of the regulatory and transcriptome carry out cross data type analyses. data sets have resulted in important Dr. Hood currently is President of the Institute for Systems Biology and is a pioneer in developing molecular and genomic instrumentation. He is a member of the National Academy of Science, the National Academy of Engineering, and the Institute of Medicine.

Dr. Hood’s research has focused on breakthroughs in biomedical science the study of molecular immunology, at the genetic level; the prestigious biotechnology, and genomics. His pro- 2004 Biotechnology Heritage Award; fessional career began at Caltech where the esteemed 2003 Association for he and his colleagues pioneered four Molecular Pathology (AMP) Award for instruments—the DNA gene sequencer Excellence in Molecular Diagnostics; and synthesizer, and the protein synthe- and the 2003 Lemelson–MIT Prize for sizer and sequencer—which comprise Innovation and Invention. He has pub- the technological foundation for con- lished more than 700 peer-reviewed temporary molecular biology. In 2000, papers and has co-authored textbooks he co-founded the Institute for Systems in biochemistry, immunology, molecular Leroy Hood, Biology in Seattle, Washington to pio- biology, and genetics. Dr. Hood holds md, phd neer systems approaches to biology and 36 patents and has also played a role in medicine. founding more than 13 biotechnology Institute for Systems Biology, Most recently, Dr. Hood was elected companies, including Amgen, Applied Seattle, WA to the Inventors Hall of Fame for the Biosystems, Systemix, Darwin, Rosetta, automated DNA sequencer. In addi- and the newly formed Hood diagnostic tion, he has received the 2006 Heinz company, Integrated Diagnostics. He is Award in Technology, the Economy currently pioneering systems medicine and Employment for his extraordinary and the systems approach to disease. A Systems Approach to and medicine over the next 10 years— Disease, the Emergence of e.g., next generation DNA sequencing and its applications to human genome Transformational Technolo- sequencing, targeted mass spectrom- gies, the Challenge of Big etry, microfluidic protein chips, new Data and Proactive approaches to protein-capture agents, P4 Medicine single-cell analyses and the use of induced pluripotential (iPS) cells to I will focus on our efforts at taking a enlighten our understand of develop- systems (medicine) approach to brain ment, disease mechanisms and disease diseases—looking at a neurodegenera- stratification. These technologies gen- tive (prion) disease, PTSD and as well erate “big data sets” with their atten- as glioblastoma and PTSD in mice and dant signal to noise challenges. humans. The mouse models allow us to Three converging opportunities—sys- analyze the initiation and progression tems medicine, big data and patient- of these diseases dynamically—enabling activated social networks—will lead to us to building models that explain the a proactive P4 medicine that is pre- pathophysiology of the disease. We dictive, personalized, preventive and have also taken a systems approach to participatory (P4). I will describe the blood diagnostics and have generated nature of P4 medicine and its societal powerful diagnostic markers for a vari- implications for healthcare. I will also ety of diseases. briefly mention some of the strategic I will then discuss the emerging tech- partnerships that we have developed to nologies that will transform biology bring P4 medicine to patients. Dr. Curtis Huttenhower is an Associate Professor in the Department of Biostatistics at the Harvard School of Public Health. He received his Ph.D. from Princeton University in the lab of Dr. Olga Troyanskaya, where he also performed his postdoctoral research at the Lewis-Sigler Institute.

his lab participated extensively in the career scientists. This research includes NIH Human Microbiome Project and a particular focus on metagenomic develops computational methods for and metatranscriptomic analyses, the functional characterization of micro- human microbiome in autoimmune dis- bial communities. His work in the area ease such as IBD, and its potential as a was recognized by an NSF PECASE diagnostic tool and point of therapeutic award, the highest honor bestowed by intervention. the United States government on early Curtis Huttenhower, phd Harvard School of Public Health, Boston, MA Bug Bytes: Bioinformatics recently become well-described using for the Human Microbiome in high-throughput sequencing, and under Health and Disease standing its role in health and disease requires techniques from genomics, Among many surprising insights, computational 'big data' mining, the genomic revolution has helped us and molecular epidemiology. I will dis- to realize that we're never alone and, cuss emerging trends in the study of in fact, barely human. For most of our the human microbiome, including lives, we share our bodies with some metagenomic and metatranscriptomic ten times as many microbes as human sequencing for mixed microbial com- cells. They are resident in our gut and on munities, experimental design for pop- nearly every body surface, and they are ulation-scale microbiome studies, and responsible for a tremendous diversity biomarker discovery for human disease. of metabolic activity and immunomodu- This will include comments on training lation. These microbial communities resources for future work in the micro- are a normal part of human health, and biome and ways in which we are begin- their disruption has been implicated in ning to identify the human and microbial diseases from colorectal cancer to pathways disrupted in inflammatory inflammatory bowel disease. conditions such as Crohn's disease and The human microbiome has only ulcerative colitis.

POSTER PRESENTATIONS Assessing Impact of Interferon Regulatory Factor - 1 on Regulatory T Cell Development and Function Abdirahman Abdullahi1, Genevieve Boily-Larouche1, Aida Sivro1, Ruey-Chyi Su1, T. Blake Ball1,2, Keith R. Fowke1,2

1Department of Introduction: Recent research of a cohort of HIV exposed sero-negative (HESN) female commercial sex Medical Microbiology, workers in Nairobi has revealed an Immune Quiescent phenotype, resulting in the potential of less HIV target cells. HESN women have an overall reduced baseline immune state, characterized by reduced T cell University of Manitoba, activation and higher regulatory T cells (Tregs) in peripheral blood. HESN women also express lower levels Winnipeg, MB, of interferon regulatory factor-1 (IRF-1) and have altered epigenetic regulation of IRF-1 responses. IRF-1, a 2University of Nairobi, critical transcription factor involved in regulating the inflammatory response, has been shown in to repress Treg development and function by directly binding and silencing the FOXP3 promoter and by modulating Nairobi, Kenya dendritic cells to favor Treg development. Methods: Full-length IRF-1 plasmid construct, driven by a CMV promoter or siRNA specific for IRF-1 will be expressed in CD4+CD25- T cells. Peripheral Tregs, CD4+CD25+FOXP3+, will be induced from transfected CD4+CD25- T cells with TGFb-1 and will be quantified by flow cytometry. Treg function will be assessed by examining Treg production of anti-inflammatory cytokines IL-10, TGF-b and suppression of proliferation of target cells. Results: We have shown IRF-1 overexpression in CD4+ T cells using the IRF-1 plasmid construct and IRF-1 silencing via siRNA in CD4+ T cells. Optimization of peripheral Treg induction assay is ongoing. Discussion: Tregs are important regulators of the human immune system; suppressing self-reactive responses and controlling systemic immune activation. Based on what we have learned from HESN subjects and from IRF-1 studies in mice, we expect that reducing IRF-1 expression will enhance Treg development and function—thereby contributing to reduced T cell activation of HIV target cells. Conversely, IRF-1 overexpression will result in decreased Treg development and Tregs with poor suppressive function. These results will provide insights on the mechanisms involved in Immune Quiescence in HESN women. Natural Mucosal Proteins Inhibit HIV-1 Infection Lindsay Aboud, Frank Plummer, T. Blake Ball, Adam Burgener

Department of Medical Background: Greater than 70% of the current 34 million people living with HIV-1 are young women, Microbiology, aged 15-24 years, who have acquired the virus through heterosexual contact. The risk of becoming infected is significantly influenced by innate and adaptive immune factors that are naturally present University of Manitoba, within the cervix/vagina, the first portal of entry for HIV-1 virus. A group of HIV-highly exposed sero- Winnipeg, MB negative (HESN) women within a sex worker cohort in Nairobi, Kenya have shown numerous differences in their immune responses and protein production, in the genital mucosal compartment, compared to women who are susceptible to infection. Immune factors that are up-regulated within the cervicovaginal compartment of HESN women include HIV-1 specific cellular CD4+ and COB+ T cell responses, HIV-specific lgA, anti-HIV factors such as RANTES, cc-chemokines, SLPI, and MIP-1 a/B, as well as various cysteine and serine antiproteases, such as Cystatin A and Serpin A1, respectively. Hypothesis: We hypothesize that specific up-regulated innate factors present within the genital secre- tions of HESN women, play a critical role in their resistance to HIV infection by protecting against mucosal inflammation/immune activation and/or through direct interference with efficientHIV-1 infection of target cells. Results: Preliminary experiments employed a TZM-bl reporter cell line, with both an R-tropic and X-tropic virus. Results confirm that specific antiproteases, including Serpin A1, C1 and Cystatin A demonstrate inhibitory activity against HIV-1 infection. Preliminary results also uncovered strong inhibitory activity (~60% inhibition) for two additional innate factors, which has not previously been observed in literature. Conclusion: These experiments have demonstrated an inhibitory role for various innate factors, including specific antiproteases, in HIV infection. These proteins are natural constituents of the female genital tract, making them ideal candidates for future microbicides. The use of natural anti-inflammatoryfac - tors In preventative strategies for HIV infection is ideal, it allows for protection against HIV, while preventing initiation of strong immune responses, and thus, subsequently, preventing influx to the site of infection of additional target cells for the virus. Does Your Neighborhood Affect Your Health and Well-Being? Sneha Abraham1, Verena Menec1,2

1Community Health Objectives: To examine whether neighborhood characteristics have differential effect on health and well- Sciences, 2Centre on being and does it differ by age. Approach: The study was based on 979 individuals aged 45 or over who were interviewed in person in Aging, University of 2007/08 as part of a longitudinal health promotion study that commenced in 1998 in a mid-Western Cana- Manitoba, Winnipeg, dian city. In the interviews, participants were asked a wide range of questions about their health, well-being, MB and health related behaviors and neighborhood perceptions. Multilevel regression analyses were conducted to examine the perceptions of neighborhood characteristics (e.g., traffic, safety concerns, and sidewalks) and their interaction with age, on outcome measures such as neighborhood satisfaction, life satisfaction, mental health and self-rated health. Results: People living in newer dwellings had statistically significantly higher scores on neighborhood satisfaction and self-rated health than people living in older dwellings (P<0.05). Traffic in the neighborhood significantly contributed to lower scores on all outcomes (P<0.001) except life satisfaction. Significant interactions emerged between safety concerns and age on three of the four outcome variables. Safety concerns did not affect older adults (65+years); however, among younger adults (45-64 years), those who reported greater safety concerns had significantly lower scores on life satisfaction, mental health and self-rated health (p<0.001 and p<0.01 resp.) Perceptions of sidewalks were not a significant predictor of any of the outcomes. Conclusion: The findings add to the growing literature on effects of neighborhood environment on health and well-being. The findings also highlight that the interaction between neighborhood characteristics and age is complex. Design and Generation of Antibodies as Therapeutics for Blocking Innate Imatinib Resistant in Chronic Myelogenous Leukemia Mahsa Abrishami, John F. DeCoteau and C. Ronald Geyer

Department of Health Given that some cancer therapeutics ultimately fail in the long-term, there is a constant demand to develop Science, Faculty of more efficient and specific treatments. Imatinib is currently the front-line therapy for chronic myelogenous leukemia (CML), which is caused by the BCR-ABL oncogene. However, Imatinib does not completely eradi- Medicine, University of cate CML stem cells, which leads to drug resistance and disease relapse. Our previous findings showed that Saskatchewan, innate Imatinib drug resistance is due to the suppression of Imatinib activity by Bone Marrow Stromal Cell Saskatoon, SK Factors, allowing leukemic cells to survive in the presence of Imatinib. In particular, our results revealed that Interleukin-3 (IL-3) and Granulocyte-Monocyte Colony Stimulating Factor (GMCSF) suppressed Imatinib activity as assessed by cell viability, Annexin V Apoptotic detection, and colony forming assays. These cytokines exert their biological activity through heterodimeric receptors with a specific alpha subunit and the signal transducing common beta subunit, a component of the high-affinity receptors for both IL-3 and GMCSF receptors. Generation of antibodies against these receptors, particularly their common beta subunit, may disrupt IL-3 and GMCSF function. The objective of this study is to develop new antibody-based drugs to disrupt IL-3R-alpha, GMCSF-R-alpha, and IL-3R-beta function. We anticipate that these antibodies will be used alone, in combination, or as co-drugs with Imatinib to eradicate CML stem cells. In order to generate the new antibodies, we used Phage-display to select and affinity-mature specific antibodies that bind to IL-3 and GMCSF receptors. Following several rounds of phage-display, antibodies that bind IL-3R and GMCSFR were identified with nanomolar dissociation constants. Taken together, all of the newly created lead antibodies successfully bound IL-3R and GMCSFR alpha and beta subunits in cell lines over expressing these receptors. Going forward, these antibodies will be tested for the inhibitory function and possible synergism with Imatinib in vitro and in vivo. This study may offer a new approach in design and generation of new therapies to eradicate the CML cells responsible for drug resistance. Protein Expression Changes of Nerve Growth Factor (NGF) During the Inflammatory Pre-Demyelinating Stages of Experimental Autoimmune Encephalomyelitis (EAE): Implications in Multiple Sclerosis (MS) Crystal May R. Acosta1, Claudia Cortes1 and Michael P. Namaka1,2,3

1Faculty of Pharmacy, Introduction: Multiple sclerosis (MS) is a disease of the central nervous system characterized by inflam- University of Manitoba, mation, demyelination, and neuro-degeneration. Studies involving nerve growth factor (NGF) have gained considerable interest due to its ability to reduce immune-mediated myelin damage. NGF exists as two pre- 2 Faculty of Medicine, dominant isoforms: proNGF (pNGF, 25 and 32 kDa) and mature NGF (mNGF, 13 kDa). The importance of the University of Manitoba, biological activity of the various isoforms in regards to recovery during the inflammatory stage of disease 3Manitoba Institute of remains unknown. The aim of this study was to examine pNGF protein expression changes in the EAE rat model during the inflammatory disease phase. We hypothesize that an increase inpNGF protein in the dorsal Child Health, Winnipeg, root ganglion (DRG) and/or spinal cord (SC) correlates with neurological recovery during the inflammatory pre- MB demyelinating stage of EAE. Methods: The EAE myelin basic protein Lewis rat model was used to study changes in pNGF protein expression. Neurological disability scores (NDS) were recorded for correlation against pNGF protein. DRG and SC tissues were harvested every 3 days up to 24 days post induction (dpi). Enzyme-linked immunosorbant assay (ELISA) was performed to quantify the 25 kDa pNGF isoform. Western blot (WB) was performed to quantify pNGF isoforms. Results: EAE NDS peaked 13 dpi. Animals recovered by 18 dpi. EAE DRG ELISA identified a significant increase in 25 kDa pNGF 21 dpi that could not be linked to neurological recovery. EAE SC ELISA identified a significant increase in 25 kDa pNGF 12 dpi that correlated with peak NDS. EAE DRG WB analysis revealed no specific changes that could be linked to neurological recovery. EAE SC WB analysis revealed an increase in 25 kDa pNGF 15-18 dpi and 37 kDa pNGF 6-9 dpi that correlates with neurological recovery. Conclusions: This research confirms the importance of the biological activity of the 25 kDa pNGF isoform in neurological recovery during the inflammatory disease stage. We believe the elevated SC 37 kDa pNGF isoform at 6-9 dpi is cleaved to produce the elevation of biologically active 25 kDa pNGF isoform seen 12 dpi. DRG pNGF isoforms do not appear to be involved in repairing EAE neurological deficits. Diverse Etiology of Osteopenia of Prematurity: From Risk Factors in the Community to the Risk Factors in NICUs Ebtihal Ali, Depeng Jiang

Department of Background: Premature infants are at risk to have bone rarefaction. Community Health Objectives: The primary objective of this study is to define the preventable risk factors that precipitate and/ or cause the rarefactions and /or fractures in premature babies in NICU in Manitoba. Sciences, University of Methods: This is a retrospective case-control study conducted at Health Sciences Center, Winnipeg, Mani- Manitoba, Winnipeg, toba. As a pilot study we will include 45 cases of osteopenia and 45 controls. The information collected MB includes: Antenatal Data: Maternal age, parity, inter pregnancy interval, ethnic background, residence in a northern community. Infant Data: Gestational age at birth, gender, birth weight. Medications such as caf- feine, Phenobarbital, diuretics, corticosteroids and their duration of treatment will be recorded. Ventilatory support type and duration. Nutritional status indicators including caloric intake and the amount of calcium and phosphate will be plotted weekly. Vitamin D intake either supplemented or included in the feeding or parenteral nutrition will be shown separately. Laboratory data: include serum calcium, phosphate, and liver function, on biweekly basis. Radiological data: the radiological data (x-rays) will be interpreted by Pediatric radiologist, who does not know about the patient data, on weekly basis. Progress: The ethical approval was obtained from the local Research Ethics Board at University of Manitoba, 2011. Significance and Future Directions: There are no reports about the prevalence of either bone rarefaction in preterm infants in Manitoba. Providing comprehensive health care will improve the bone status of these premature babies in infancy and childhood, lessen the morbidity associated with recurrent fractures, poor oral health and the burden on the health care system. We are hoping to introduce the usage of the Quanti- tative ultrasonography (QUS) of bone and use it as a screening tool to identify the infants with osteopenia. Chondroadherin Fragmentation as a Biochemical Marker for Early Stage Disc Degeneration Bashar Alkhatib, Patrik Onnerfjord, Rahhul Gawri, Jean Ouellet, Peter Jarzem, Dick Heinegard, John Mort, Peter Roughley and Lisbet Haglund

Division of Disc degeneration has been strongly associated with back pain. At present, little is known about the Experimental Surgery, molecular mechanisms involved in the degeneration of intervertebral discs and how these may differ from normal turnover of the tissue. As a result of this, a biomarker for disc degeneration has not yet been McGill University, identified and we propose chondroadherin(CHAD) fragmentation as a potential marker. The aims of this Montreal, QC study were to determine whether CHAD fragmentation is unique to disc degeneration, to characterize the cleavage site within CHAD at which fragmentation occurs, and to identify the protease capable of CHAD cleavage at the characterized site. CHAD fragmentation was studied using SDS-PAGE and western blotting in combination with specific antibodies. Characterization of the cleavage site was achieved by fractionating a degenerate surgical disc sample using SDS-PAGE. Gel portions containing the CHAD fragment were excised and identified by mass spectrometry. An anti-neoepitope antibody was raised to recognize the cleavage site sequence. Protease digests of macroscopically normal disc tissue were performed with proteases known to be upregulated during disc degeneration. Digest tissue was extracted and fractionated using SDS-PAGE. lmmunobloting was performed with an anti-CHAD antiserum and the anti-neoeptiope antibody. Evidence for proteolytic degradation of CHAD was observed in adult discs showing degeneration but not in tissue from a macroscopically normal disc. Furthermore, the higher the degree of degeneration seen in the disc, the higher the amount of CHAD fragmentation. Upon analysis with the anti-neoepitope antibody, it was apparent that CHAD fragmentation occurred at the same cleavage site in degenerate discs from apparently healthy donors, surgical samples from adults with disc degeneration, and adolescents with premature degeneration due to scoliosis. Normal tissue samples showed no anti-neoepitope antibody binding, confirming that CHAD fragmentation at this site was not present in the healthy disc. A single protease was identified that was capable of CHAD cleavage at an identical site to that seen inall tissue donors with disc degeneration as confirmed by the anti-neoepitope antibody. CHAD fragmentation can be used as a biomarker to distinguish normal aging from disc degeneration. This evidence can be used to develop a potential immunoassay to screen the serum of at-risk patients and detect early disc degeneration. Examining the Explanatory Usefulness of Merton’s Theory of Unintended Consequences for Public Health Interventions Lisa K. Allen-Scott and J.M. Hatfield

Faculty of Medicine, Background: In 1936 the seminal article by Robert Merton, “The Unanticipated Consequences of Purposive University of Calgary, Social Action” was published and today remains a key source of unanticipated or unintended consequence (UC) theory. Merton concludes his paper with a call for the systematic study of mechanisms involved in Calgary, AB the development of UC in various disciplines. Although it has been over 70 years, there remains a paucity of UC theory for specific fields of study. Public health is a prime example where UC theory remains limited, despite a building body of evidence that harmful UC are occurring. This study will begin to address this gap by investigating the explanatory usefulness of Merton’s theory and underlying mechanisms of UC for Public Health Interventions (PHIs). Research Question: Does Merton’s theory and mechanisms of UC hold their explanatory usefulness for PHIs that occur in various contexts? Methods: The explanatory usefulness of Merton’s five proposed mechanisms of UC: (1) ignorance, (2) error, (3) immediate interest, (4) basic values and (5) self-defeating prophecy, will be examined using realist review methodology. The realist review will facilitate the examination of unique context (C) and mechanism (M) interactions that may be involved in the development of UC (the outcome O) in PHIs. Thematic content analysis will be used to identify emerging PHI UC mechanisms, the contexts that they occur in, and unique UC outcomes. Results: Based on a synthesis of evidence, we will identify key UC mechanisms and ultimately adapt Mer- ton’s theory for PHIs. Contradictory evidence and the role of context for the existence of PHI associated UC will be reported. Recommendations will be made to raise consciousness of the potential for anticipation of UC associated with well-meaning PHIs. Conclusion: This research will provide foundations for integration of operational theory into PHI evaluation models or health impact assessment frameworks. This integration will offer guidance for public health professionals and researchers when discussing PHI associated UC. NMDA Receptor Activation Induces Contraction and Calcium Influx in Cultured Human Airway Smooth Muscle Cells Vidyanand Anaparti1, R. Ilarraza1, A.J. Halayko1,2 and R. Moqbel1

1Departments of Background: Eosinophil-derived glutamate and kynurenines exert their function through N-methyl-D-as- 2+ Immunology and partate receptors (NMDA-R), a family of heterotetrameric ion channels permeable to Ca and comprised of NR1, NR2 and NR3 subunits. The NR1 subunit is essential for a functional NMDA-R. In animal models, NMDA- 2 Physiology, University R stimulation in the lung induces AHR and increases airway contractility. We hypothesized that NMDA-R of Manitoba, Winnipeg, subunits are expressed by HASM cells, that subunit expression is modulated by pro-inflammatory cytokine MB exposure, and activation of functional NMDA-R induces contraction. Results: While qPCR confirmed that HASM cells express majority of the NR subunits, NR1 expression was determined by WB. TNF and IL-1b @ 10ng/mL selectively increased NR1 expression in these cells. Stimula- 2+ tion with NMDA, a specific NMDA-R agonist, induced an steady increase in [Ca ]i (peak 0.6µM after ~120 seconds). Ca2+ mobilization was abrogated by D-APV, an NMDA-R antagonist (n=5). Notably, discrete myo- 2+ cyte subpopulations revealed four distinct patterns in [Ca ]i flux responses: so called “early” (~15), “phasic” (~18%), “late” (~50%), and “no” responders (~22%). TNF pre-treatment had a noticeable effect on distribution of myocytes that exhibit different temporal patterns of Ca2+ mobilization. Functional relevance for Ca2+-flux was confirmed using collagen gel assays, in which dose -dependent contraction was evident (maximum of 25% shortening with 100μM NMDA). NMDA-induce gel contraction was inhibited by D-APV antagonism (n=3). Conclusion: Taken together, our data suggests NMDA-R can participate in regulation of HASM contraction in vitro, and thus could be of relevance to AHR associated with asthma. Regulation of Wnt Signaling by Pygopus Post-Translational Modification Phillip Andrews and Kenneth Kao

Faculty of Medicine, The Wnt signaling pathway plays important roles in normal embryonic development and normal tis- Memorial University, St. sue homeostasis in adults. I t is required to induce cell differentiation and cell proliferation, the latter of which, if not tightly regulated can promote uncontrolled cell growth and lead to the development John’s, NL of cancer. Pygopus (Pygo) is a protein that has been shown to play roles in both Wnt signaling and cancer. Pygo is directly linked to the active Wnt/b-catenin transcriptional complex through binding to the adapter protein B cell lymphoma 9 (Bcl-9) and it can also interact with Histone H3 trimethylated at Lysine 4 (H3K4me3), an epigenetic mark of active transcription. In this complex, Pygo likely serves as a positive regulator of transcription by promoting euchromatin formation at the vicinity ofWnt target genes through the recruitment of histone acetyltransferases, such as members of the cyclic AMP response element binding protein (creb) binding protein (CBP)/p300 family. Here, further investigation into the role of Pygo in Wnt signaling and its binding to histone acetyltransferases revealed that Pygo itself may be a target of acetylation by CBP and p300 family members at specific Lysine residues. The observation of Pygo acetylation was also correlated with Wnt pathway activation, as well as an increased affinity for Bcl-9, as well as H3K4me3. It therefore appears that acetylation of Pygo is important in controlling Wnt target gene output and may be important in tumors arising from aberrant Wnt/b-catenin signaling. Expression Analysis of Glucose Transporter 1 Gene in HIV Highly Exposed yet Seronegative Commercial Sex Workers, Nairobi, Kenya Winnie Apidi1, Ruey Chyi Su1, Joshua Kimani1,2, Frank Plummer1, Blake Ball1

1Department of Introduction: There has been altered susceptibility to HIV-1 infection observed in multiple cohort studies es- Medical Microbiology, pecially in highly exposed commercial sex workers. Resistance to HIV-1 among commercial sex workers who are continuously exposed to HIV-1 yet remain uninfected by the virus has been reported. (Fowke, et al, 1996; University of Manitoba, Plummer, et al, 1999; Fowke, et al, 2000; Ball, et al, 2007) A gene expression analysis showed differential regula- Winnipeg, MB tion of the glycolysis/gluconeogenesis pathway in HIV-1 exposed yet seronegative CSWs. (Songok, et al, 2012) 2University of Nairobi, The first critical regulatory step in glucose metabolism is glucose entry into cells through facilitated diffusion by proteins of the glucose transporter (GLUT) family. (Frauwirth et al, 2002; Jacobs, et al, 2008) Overexpression Nairobi, Kenya of Glut1 leads to increased glucose uptake and glycolysis which is required to mount a functional immune response necessary for rapid cell growth and proliferation in T lymphocytes. This is vital in understanding how normal lymphocyte function is regulated and fuelled. T cells use glucose and glutamine as their primary fuel source. Activated T cells thus have increased metabolic requirements provided by glycolysis. (Fox et al, 2005; Maclver et al, 2008;) Methods: Study population was drawn from the Pumwani Sex Worker Cohort, Nairobi. Study groups included: HIV highly exposed yet seronegative (HESN) CSWs (>7 years); newly enrolled HIV-uninfected (<7 years); HIV-infected and lowly-exposed HIV negative antenatal clinic attendees (low risk group) (n=14each). Total RNA was extracted from PBMCs using Trizol (Invitrogen, USA), cDNA was synthesized and relative mRNA expression determined using SYBR Green by quantitative real time PCR. Results: Each assay was normalized using 18s rRNA gene. There is significant difference between highly exposed yet uninfected subjects (HESNs) and newly enrolled HIV uninfected CSWs. (p=0.0056) There is no significant difference between HESNs with the HIV negative antenatal clinic attendees (p=0.8628); and HIV infected CSWs (p=0.5399). Conclusion: Significantly lower mRNA expression of Glut1 was observed in HESNs when compared to their uninfected yet susceptible counterparts. Following studies of Glut 1 protein expression and uptake studies are underway to understand the role Glut1 in glucose metabolism in HIV resistance. A Mouse Model of Bowen-Conradi Syndrome Displays Severe Developmental Delay and Neural Tube Defects Joy Armistead1,2, Hao Ding1,2, Biswajit Chowdhury1,2, Nehal Patel1, Barbara Triggs-Raine1,2

1Department of Bowen-Conradi syndrome (BCS) is a lethal autosomal recessive disorder found in the Hutterite population, Biochemistry and with an average age at death of thirteen months. Children born with the disorder display severe developmental delay and psychomotor retardation, and die due to causes related to failure to thrive. We previously Medical Genetics, discovered that BCS is caused by an A>G mutation in EMG1, a gene which plays a role in ribosome biogen- University of Manitoba , esis. A complete deletion of EMG1 in the mouse was lethal at embryonic day (E) 3.5, precluding further study 2Manitoba Institute of or generation of EMG1-deficient cell lines. We therefore set out to create a mouse model of Bowen-Conradi syndrome by incorporating the A>G mutation in EMG1 by homologous recombination. This would allow us Child Health, Winnipeg, to examine the progression of the disease throughout development and in multiple tissues. Mice heterozy- MB gous for the mutation were obtained and were viable and fertile, however intercrossings of heterozygous mice revealed an absence of homozygous mutant offspring, suggesting prenatal lethality. Embryos were therefore examined at different stages of development, and homozygous mutants were obtained at stages E8.5-E11.5. These embryos were significantly smaller than control littermates, and approximately 50% also presented with an open cranial neural tube. These results suggest that a mouse model of BCS recapitulates the developmental delay seen in BCS patients, and that the central nervous system is a major target of the EMG1 defect. Histological studies are now in progress to compare the morphology and characteristics of the developing central nervous system in A>G homozygous embryos compared to controls. These studies will provide information that will be useful in determining the mechanism underlying BCS and in evaluating the mouse as a model for future assessment of potential therapies. Elucidation of the Regulatory Role of Scleraxis in Modulating Fibrotic Gene Expression in the Heart Rushita A. Bagchi1, Patricia Roche1, Ronen Schweitzer2, Michael P. Czubryt1

1Institute of Introduction: Cardiac fibrosis has a significant effect on patient outcomes and continues to be a major Cardiovascular clinical problem. There is a lack of effective therapies owing to incomplete understanding of the mediators involved in cardiac fibrosis and remodeling process. Our laboratory has previously demonstrated the role of Sciences, University of the bHLH transcription factor Scleraxis (Scx) as a key regulator of collagen 1a2 gene expression in cardiac Manitoba, Winnipeg, fibroblasts and myofibroblasts. Here we identify novel gene targets of the fibrotic process regulated by Scx. MB, 2Department of Methods: Cardiac ventricular fibroblasts were isolated from adult rat using Langendorff perfusion followed by enzymatic digestion. Cells at second passage were infected with adenoviral constructs encoding Scx or Cell and Developmental Smad3 or Smad7; RNA and protein was harvested 24h post-transduction and subjected to qRT-PCR analysis Biology, Oregon Health and immunoblotting respectively. Loss of function experiments were carried out using adenovirus encoding and Science University, dominant negative Scx mutant. Cardiac tissues from scleraxis knockout (Scx KO) and wildtype (WT; control) mice were harvested, total RNA and/ or protein isolated, and processed as above. Pressure-overloaded mu- Portland, OR rine hearts were also analyzed for collagen expression and tissue fibrosis. Results: Smad3 or 7 overexpression resulted in significant up-regulation or attenuation of Scx expression respectively. The expression of matrix remodeling enzymes MMPs 9 and 11, and TIMP3 decreased in response to Scx overexpression in vitro. The gene expression of proteoglycans versican and decorin were also decreased following Scx overexpression. No significant change was observed in decorin and there was a loss of versican in scleraxis KO hearts. Scx over-expression led to increase in thrombospondin 4, periostin, collagen type I (Col I) and a-smooth muscle actin (aSMA). These genes were strongly downregulated in Scx KO hearts. Increased expressions of Scx and Col I was concomitant with increased fibrosis in the hypertrophied heart. The DNA-binding domain mutant form of Scx was able to attenuate the effect of pro-fibrotic TGF-b1 on cardiac collagen expression. Conclusion: Scleraxis exerts wide-ranging effects on extracellular matrix- related gene expression as demonstrated by our experiments here. The effect on aSMA expression suggests that Scx may be a key regulator of fibroblast-to-myofibroblast phenoconversion - a process known to accompany cardiac remodeling and- fi brosis. Our findings strongly support our contention that Scx is a novel therapeutic target for cardiac fibrosis. Regulatory B cells Suppress the Progression of Fatal Autoimmunity in Lupus-Prone Mice Yuriy Baglaenko1,2, Nan-Hua Chang1, Evelyn Pau1,2, Joan Wither1,2

1Toronto Western Aim of study: Previous work from our lab has shown that the introgression of a NZB chromosome 1 (c1) Hospital, University interval extending from 135 to 179Mb onto the non-autoimmune C57BL/6 (B6) background results in increased B and T cell activation, elevated ANA levels and fatal kidney disease. Interestingly, introgression Health Network, and of an additional NZB c4 interval extending from 30 to 150Mb onto the NZB c1 mice leads to suppression of 2University of Toronto, autoimmunity with decreased renal disease and increased survival. Given the expansion of splenic CD5+ B Toronto, ON cells in suppressed mice, we sought to investigate their role in autoimmunity. Methodology: Cellular phenotypes and intracellular cytokine production were examined by flow cytometry of de novo and stimulated splenocytes, respectively. Serum levels of anti-nuclear antibodies were measured by antigen specific ELISA. Adoptive transfer experiments were performed by injecting 5-10 million B cells intravenously into c1.Thy1aIgHa recipients. Results: There was a significant increase in IL-10 producing B cells in suppressed mice with an inverse correlation between the levels of anti-ssDNA IgG and the frequency of IL-10 producing B cells. In support of a direct suppressive capacity for the expanded CD5+ B cells, adoptive transfer of total B cells from NZB c4 mice, but not B6, mice into autoimmune prone c1.Thy1aIgHa recipients reduced the proportion of activated B cells, germinal centre B cells, memory/effector T cells, and IFN-γ or IL-17-secreting T cells. Conclusions: Taken together, these data indicate that the expansion of CD5+ IL-10 producing B cells can suppress the progression of fatal autoimmunity. Using Structure-Guided Mutagenesis to Selectively Decouple Deubiquitinating Activity from the Polyprotein Processing Activity of the Equine Arteritis Virus Papain-like Protease 2 Ben A. Bailey-Elkin2, Puck B. van Kasteren1, Terrence W. James2, Dennis K. Ninaber1, Corrine Beugeling1, Mazdak Khajehpour3, Eric J. Snijder1, Brian L. Mark2, Marjolein Kikkert1

1Leiden University Protein ubiquitination regulates important innate immune responses. Ubiquitin (Ub) can be attached to Medical Center, Medical lysine residues on cellular proteins to promote, among other activities, the innate immune responses of the cell. These pathways can in turn be downregulated by the removal of Ub from cellular proteins by deubiqui- Microbiology, Leiden, tinases (DUBs). Netherlands, Arteriviruses are positive-sense RNA viruses which share a common mechanism of gene expression, 2Department of whereby a number of viral nonstructural proteins (nsps) are initially expressed as a single polyprotein, which is then cleaved into functional units by papain-like protease (PLP) domains. Equine arteritis virus (EAV) Microbiology, University posses a PLP2 domain at the N-terminus of nsp2, which not only cleaves within the viral polyprotein but of Manitoba, also suppresses the host innate immune response by removing Ub from cellular proteins. The discovery of 3Department of virus-encoded DUBs suggests they remove Ub to evade Ub-dependent anti-viral responses; however, this has never been conclusively demonstrated in virus-infected cells. Chemistry, University of In order to dissect the dual specificity of EAV PLP2, which relies on a single catalytic active site, we deter- Manitoba, Winnipeg, mined the crystal structure of PLP2 in complex with Ub (1.45 Å). PLP2 binds Ub using a zinc finger that is MB uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc- dependent OTU DUBs. Notably, the Ub-binding surface is distant from the catalytic active site, which allowed us to mutate key residues on this surface that were found to interact directly with Ub during binding. These mutations were found to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, however the loss of DUB activity strikingly enhanced innate immune signaling. Com- pared with WT virus infection, IFN-b mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in ar- teriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual specificity proteases. The Relationship Between Immunization use, Birth Parity, and Gender in Northern Karnataka, India Dana Baker and James Blanchard

Department of Rationale: As communicable diseases are a serious burden to healthcare systems, immunizations serve as Community Health important forms of protection and prevention particularly for newborns, infants and children. However, im- poverished rural regions in developing countries such as India experience higher rates of non-immunization Sciences, University of among children, which contributes to increased child morbidity and mortality rates. Manitoba, Winnipeg, Objective: To better understand factors associated with early child immunization statuses, this study will MB investigate the relationship between immunization status, birth parity and gender in children between birth and 2 years living in 8 of the poorest districts of northern Karnataka, India. Regional variation in morbidity and mortality rates will be investigated in relation to immunization. Methods: Secondary data from the Sukshema Project will be utilized for this study. Data was collected over a 12-month period from 8 of the poorest districts within rural northern Karnataka, India. The total sample population was n = 23,998 and was based on a systematic stratified random design. Predictor and outcome variables include child’s gender, birth parity, and immunization status. Variables at the family level including, age of parents, annual household income, highest level of education attained and family values and beliefs will also be included and controlled for to address any potential underlying correlates. SPSS and other appropriate statistical analysis software (e.g., SAS) will be utilized to calculate basic descriptive statistics and logistic regression odds ratio models. Results: Pending. Conclusions: This research will inform knowledge mobilization efforts seeking to improve early immunization interventions directed at women and families who are planning on having children and/or who are expecting a child. Healthcare professionals and policy makers will gain insight as to where immunization resources, program planning, and funding should be allocated. Additionally, this study will inform new research to increase understanding of the broader issues surrounding neonatal and child morbidity and mortality rates in these areas. Exploring Health Risk Behaviour among Inner City Youth: A Mixed Methods Approach Shivoan Balakumar

Department of Background: Youth belonging to low income families or living in inner-city, low income neighborhoods Community Health have been shown to be at higher risk for engaging in negative health behaviours, persisting with those behaviours, and ultimately suffering from downstream, chronic health consequences. Comprehensive youth Sciences, University of risk behaviour studies and prevention efforts have traditionally involved large population-based surveys, Manitoba, Winnipeg, producing behavioural prevalence rates and identifying potential risk factors. Few studies have explored the MB phenomena of youth engagement in health risk behaviour from a youth-centered perspective. Objective: This study aims to shed light on the perspectives of inner-city Winnipeg youth about their engagement in a variety of health related behaviours; specifically, whether youth are concerned about their engagement in any particular risk behaviours, what types of barriers prevent them from changing these behaviours, and what types of interventions might be useful in supporting their decision to change. Methods: We will use a two phase sequential, mixed methods design. Phase 1 will involve a secondary analysis of a survey (n-250) of youth (14-24 yrs) from Winnipeg’s inner city. Data from these surveys will provide demographic information, the prevalence of youth engagement in health risk behaviours, and finally, whether youth are concerned about their engagement in particular risk behaviours. Phase 2 will involve age and gender stratified focus groups of participants from Phase 1. Results from Phase 1 will inform which risk behaviours to explore further in the Phase 2 discussions. Qualitative analysis of the Phase 2 data will explain and supplement Phase 1 quantitative data. Results: Pending Fall/2013 Significance: This study will help inform strategic behaviour interventions targeted towards increasing youth capacity to change behaviours that are of primary concern to them. By aiding youth in their efforts to change behaviours that they already have an interest in changing, we may be able to measurably improve their self efficacy, and eventually increase their concern for other negative health related behaviours. Modulation of OVA-Induced Airway Hyperresponsiveness and Inflammation by Pentraxin 3 (PTX3) in a Murine Model Jyoti Balhara, Lianyu Shan, Abdelilah Soussi Gounni

Department of Introduction: Pentraxin-3 (PTX3) is a member of the long pentraxins family. It activates the innate and Immunology, Faculty of adaptive immune systems, playing an important role in providing immunity against various pulmonary infections. Considering its role in fostering lung immunity, we examined the role of PTX3 in asthma. Medicine, University of Objective: We have recently demonstrated an enhanced expression of PTX3 in bronchial biopsies of allergic Manitoba, Winnipeg, asthmatics. Additionally we found enhanced levels of PTX3 in BALF from asthmatic subjects as compared MB to their healthy counterparts. Thus, in this study we wanted to determine the effect of PTX3 deficiency in a murine model of OVA-induced asthma. Results: Wild type (WT) and PTX3 Knockout (KO) mice were sensitized and challenged with OVA. Sensitized PTX3 KO mice exhibited an enhanced airway and tissue resistance in response to methacholine (MCh) in contrast to their WT counterparts. We observed an increase in the infiltration of inflammatory cells in BALF obtained from PTX3 KO mice upon OVA sensitization/challenge as compared to their WT littermates. However peribronchial and perivascular inflammation and goblet cells hyperplasia showed no significant difference in both mouse strains. Further we found an enhanced induction of IL-4, IFN-gamma, IL-17 and IL-10 production in the lungs of PTX3 KO mice as compared to WT mice upon OVA challenge. In lymph node, IL-17A recall response was found to be greater in PTX3 KO mice than WT mice but no difference was observed in term of IL-4, IFNgamma and IL-10 production. Conclusion: Taken together, we conclude that lack of PTX3 predisposes mice to airway hyperresponsiveness and an enhanced inflammation. Degradation of Postural Control with Age Jennifer Baltich, Vinzenz Von Tscharner, Benno M. Nigg

Faculty of Kinesiology, Introduction: It is believed that aging negatively impacts the ability of the body to maintain postural stabil- University of Calgary, ity due to degraded control systems. Postural control is often assessed using center of pressure (COP) movement magnitude measures. However, it is important to investigate the temporal aspects of the COP signal as Calgary, MB it may provide information about the underlying control mechanisms. Therefore, the purpose of this study was to quantify the effects of aging on the magnitude and temporal structure of the COP signal. Methods: Ground reaction force data was collected from 48 subjects (24 elderly, 24 young) as they completed single limb stance trials. The regularity of the COP signal was quantified by calculating the sample entropy (SampEn) for consecutive gradual randomizations of the original COP signal. The entropic half-life E(1/2) was identified as the time at which the SampEn transitions from being more regular to more irregular (SampEn = 0.5). The magnitude of COP movement was quantified using the path length and the 95% ellipse area. Results: There was no significant difference between the elderly and the young subjects for the E(1/2) in the medio-lateral direction. However, the elderly demonstrated a significantly shorter (p<0.001) E(1/2) in the AP direction (mean (SD) - 78 (16) ms) compared to the younger subjects (mean (SD) - 139 (40) ms). The elderly subjects also demonstrated a longer path length (mean (SD) - 1573 (322) mm) compared to the younger subjects (mean (SD) - 1095 (258) mm) and a larger 95% ellipse area (mean (SD) - 889 (281) mm2) compared to the younger subjects (mean (SD) - 509 (191) mm2). Conclusion: The results from this study demonstrated the reduction in the E(1/2) in conjunction with an increase in the movement magnitude of the COP with aging. Even though elderly subjects demonstrated more frequent postural adjustments, this did not help to reduce their magnitude of movement. Degradation of the postural control system with aging may inhibit the ability of elderly subjects to detect and control changes in the COP position, similar to deficits found in postural control following injury. Decreased Expression of the Deleted in Liver Cancer 1 (DLC-1) Tumor Suppressor Affects Mammary Gland Development and Cell Polarity Pratima Basak1,2,3, Rachelle Dillion1, Heather Leslie1, Afshin Raouf1,2, Michael R.A. Mowat1,3

1Manitoba Institute of Introduction: The Deleted in liver cancer 1 (DLC1) gene is a tumor suppressor, which maps to human chro- Cell Biology, CancerCare mosome 8p21-22. It was originally discovered as a site of frequent deletions in primary hepatocellular carcinoma. DLC1 is a multiple-domain RhoGAP protein. It suppresses cell proliferation, triggers apoptosis and Manitoba, reduces migration and invasion when reintroduced into DLC1 deficient tumor cell lines. DLC1 undergoes 2Regenerative Medicine heterozygous deletion in over 50% of breast, lung, prostate and colon cancers. Increased Promoter DNA Program, 3Department methylation is frequently seen in several tumor types, which silences the other allele. Transfection of DLC-1 into deficient breast cancer cells prevents tumor formation after inoculation into immunocompromised of Biochemistry and nude mice. Homozygous loss of DLC1 results in an embryonic lethal phenotype in mice. In this study, we Medical Genetics, wanted to learn if DLC1 plays a role in normal mammary gland morphogenesis and epithelial cell polarity. University of Manitoba, Methods & Results: To learn if DLC1 affects mammary gland development, whole gland mount preparations were prepared. Our preliminary data suggests that DLC1 heterozygous gene-trapped mice showed Winnipeg, MB abnormalities in mammary gland development. These included increased ductal branching and deformi- ties of the terminal end buds. Mammary glands from DLC1+/- mice also showed a thickened stroma layer with increased collagen deposition, as determined by Masson’s Trichrome staining, when compared with WT mice. Interestingly when primary mammary cells were plated in 3D matrigel cultures, the DLC1+/- cells formed solid spheres in contrast to WT cells, which formed hollow acini structures. To learn the effects of complete DLC1 loss, the primary wild type mammary cells were infected with shRNA expressing lentiviral vectors. We observed that decreased DLC1 transcript levels in the wild type mammary cells resulted in formation of solid acini structures, which was similar to the heterozygous DLC1+/- cells. The scrambled shRNA controls showed hollow acini structures. Conclusion: These results indicate that reduced DLC1 expression disturbs the normal polarization and lumen formation that occurs during epithelial cell morphogenesis. This suggests that DLC1 normally functions to maintain mammary epithelial cell polarity. These results also suggest that DLC1 is haploinsufficient. Sleep Architecture in Ziprasidone-Treated Bipolar Depression: A Pilot Study Anusha Baskaran1,2, Dave Summers1,2, Stephanie L.M. Willing2, Ruzica Jokic1,2, Roumen Milev1,2

1Centre for Introduction: This study investigated the effect of ziprasidone augmentation therapy on sleep architecture Neuroscience Studies, in bipolar depression. Methods: We conducted a double-blind, randomized, placebo-controlled clinical pilot trial of ziprasidone 2 Department of versus placebo in Diagnostic and Statistical Manual of Mental Disorders, fourth edition bipolar disorder with Psychiatry, Queen’s current major depressive episode. The effects during acute (2–5 days) and continuation treatment (28–31 University, Kingston, ON days) were measured. Main outcomes were sleep architecture variables including rapid eye movement sleep (REM) and slow wave sleep (SWS) measured by polysomnography. Secondary outcomes included subjec- tive sleep quality measures and illness severity measures including the 17-item Hamilton Depression Rating Scale (HAMD-17), Montgomery Asberg Depression Rating Scale (MADRS), Hamilton Anxiety Rating Scale (HAMA) and Clinical Global Illness Severity (CGI-S) scores. Results: The completer analysis comprised of 14 patients (ziprasidone, N = 8 and placebo, N = 6). Latency to REM, duration of SWS, duration of stage 2 sleep, total sleep time, onset to sleep latency, number of awaken- ings and overall sleep efficiency significantly improved in ziprasidone-treated participants over placebo. CGI-S and HAMA scores also significantly improved. No significant difference between treatment groups was seen on the HAMD-17, MADRS or in self-reported sleep quality. Increase in SWS duration significantly correlated with improvement in CGI-S, however, this finding did not withstand Bonferroni correction. Conclusion: Adjunctive ziprasidone treatment alters sleep architecture in patients with bipolar depression, which may partially explain its mechanism of action and merits further investigation. Salicylate-Based Non-Steroidal Anti-Inflammatory Drugs (NSAIDs): Novel Catalytic Inhibitors of Human Topoisomerase Iiα Jason T. Bau, Ebba U. Kurz

Southern Alberta Topoisomerase IIα (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regu- Cancer Research lating DNA topology, transcription and sister chromatid separation prior to mitosis. Topo II is also the intracellular target of a number of commonly used cancer chemotherapeutic agents (topo II poisons), treatment Institute, Faculty of with which results in the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, Medicine, University of non-DNA break inducing topo II catalytic inhibitors have also been described and have more limited use in Calgary, Calgary, AB clinical chemotherapy. These agents, however, may alter the efficacy of treatment regimens incorporating topo II poisons, such as doxorubicin and etoposide. We have identified salicylate, the primary metabolite of aspirin, and other salicylate-based NSAIDs as novel catalytic inhibitors of topo II. We have demonstrated that brief pretreatment of human breast cancer cells with these compounds attenuates the cytotoxicity of the topo II poisons doxorubicin and etoposide. This is a direct consequence of topo II catalytic inhibition and is independent of salicylate’s previously characterized roles as an inhibitor of cyclooxygenases and NF-κB. Importantly, salicylate and related compounds attenuate the formation of topo II-linked DNA complexes, thereby preventing the accumulation of DNA double-stranded breaks. By examining each step of the topo II catalytic cycle, we have now identified the biochemical mechanism of topo II inhibition by salicylate. Salicylate-based NSAIDs are unable to intercalate DNA, do not prevent topo II-DNA binding and do not promote stabilization of topo II-DNA closed clamps. Interestingly, these compounds decrease topo II ATPase activity. This inhibition is non-competitive, a mechanism that is distinct from previously characterized inhibitors of topo II ATPase activity. In silico analysis predicts that this inhibition is the result of salicylate binding within ATP-binding pocket of topo II. Given the widespread use of aspirin and salicylate-based NSAIDs, including among those diagnosed with cancer, our observations will inform animal-based studies investigating the impact of salicylate co-administration on chemotherapeutic efficacy. Regulation of Adult Neurogenesis in the Mouse Eye in Vivo Marie-Claude Bélanger1,2 and Michel Cayouette1,2,3

1Institut de Recherches Introduction: Fish and amphibian retinas have the capacity to grow throughout life and regenerate follow- Cliniques de Montréal, ing injury. These features are mainly attributable to Müller glia, which can de-differentiate to give rise to new glial cell or neurons, or to Retinal Stem Cells (RSCs) of the ciliary margin zone (CMZ). In mammals, 2 Division of however, these self-repair mechanisms are limited, as Müller glia have a very low propensity for de-differ- Experimental Medicine, entiation, and RSCs, although able to generate neurons in culture, remain quiescent in vivo. These observa- McGill University, tions raise the question: which mechanism prevents adult mammalian neurogenesis in vivo? Methods: Considering the importance of Notch signaling in the regulation of proliferation and differentia- 3Département de tion of various stem cells/progenitor population during development, we reasoned that regulators of the Médecine, Université de Notch pathway might play part in neurogenic activity in adult tissue. More specifically, we focus on the Montréal, Montréal, QC endocytic adaptor protein Numb, an essential cell-fate determinant during retinal development, which antagonises Notch signaling. Our analysis reveals that Numb is expressed in the mouse retina and Ciliary Body (CB) from embryogenesis through adulthood, suggesting a role for Numb in adult tissue homeostasis. To test this hypothesis, we generated conditional mouse mutant depleting the protein Numb specifically in the retina and CB. Results: Interestingly, we found that loss of Numb function resulted in a massive cellular expansion in the adult eye, which appeared to contain several types of differentiated neurons. In addition, in the neurosphere assay, spheres lacking Numb are significantly bigger in size, suggesting a role for Numb in repressing the proliferation capacity of adult RSCs. Conclusion: Together, these preliminary results suggest that Numb may have a continuing role in the maintenance and regulation of adult stem cell proliferation. Future experiments will aim to unravel the molecular mechanism by which Numb inhibits Retinal Stem Cells proliferation in vivo. Investigating Solvent Use and HIV in Manitoba Courtney Bell1,2, Margaret Ormond2, Chelsea Jalloh1,2, John Wylie1, Javier Mignone1, Keith Fowke1

1Department of Individuals that work closely with the Winnipeg solvent-using population in an outreach/support capacity Medical Microbiology, have observed that HIV is present within the solvent-using population. Solvent use is a poorly understood and understudied substance use issue associated with poverty that occurs worldwide. Solvents are a type of University of Manitoba, inhalant and include many common chemicals such as gasoline and lacquer thinner, which have psychoac- 2Sunshine House, tive effects when inhaled. Manitoba appears to have a population of solvent users that are demographically Winnipeg, MB unique from solvent-using populations described in the limited, existing literature. Clinicians have provided anecdotal evidence that suggests solvent use damages gastrointestinal and respiratory epithelium. Since HIV disrupts the integrity of mucosal barriers and induces a state of chronic immune activation, this may have important consequences for solvent users in terms of HIV susceptibility and disease progression. This research aimed to investigate both social and biological aspects related to solvent use that may be relevant for HIV through an interdisciplinary approach adopting methodologies from community based research and basic science. The first phase of this project involved partnering with a community-based organization in Winnipeg, Sunshine House, whose client base includes solvent users. Three focus groups and ten qualitative individual interviews with solvent users were carried out as well as 8 interviews with key informants. The purpose of these consultations was to investigate characteristics of the population regarding solvent use and inquire about willingness to participate in future research. Common themes identified include the use of solvents both recreationally and as a coping mechanism, the importance of relationships, the willingness to participate in research, the obstacles experienced related to solvent use, and the need for a treatment option targeted towards the population’s specific needs. The next phase of this research involves obtaining biological samples and exploring the impact of solvent use on the immune system. Proteomic Analysis of Cervicovaginal Fluid Uncovers Immune Pathway Variation Between the Follicular and Luteal Phases of the Menstrual Cycle - Implications for HIV Susceptibility Kenzie M. Birse1,2, Richard M. Novak4, Garrett R. Westmacott3, Stuart McCorrister3, T. Blake Ball1,2,5, Adam Burgener1,2

1Department of Introduction: HIV infection occurs more frequently during specific stages of the female menstrual cycle as Medical Microbiology, shown in animal and tissue models. The reason for this is unknown, but it is thought to be due to changes in the immunological environment. Sex hormones, such as estrogen and progesterone, have been shown to University of Manitoba, affect immune factor expression, where higher estrogen promotes heightened immunity and progesterone 2Proteomics Group, reduces certain immune functions to allow implantation of a potential embryo. Immune factor expression National Lab for HIV in the female genital tract may affect HIV susceptibility, as several soluble innate factors have been shown to inhibit HIV in vitro. This study uses proteomics to compare the immune mediators expressed during the Immunology, 3Mass follicular and luteal phases of the menstrual cycle to better understand these immunological changes and Spectrometry Core their implications for HIV susceptibility. Facility, NML, PHAC, Methods: Cervicovaginal lavage was collected from 37 healthy women and analyzed by tandem-mass spectrometry. Samples were grouped based on days since last menstrual period into either the follicular Winnipeg, MB, phase (days 7-10) or luteal phase (days 20-25) of the menstrual cycle. Data was analyzed by a combination of 4 University of Illinois, differential protein expression analysis, hierarchical clustering and pathway analysis. Chicago, IL, 5University Results: Of the 617 proteins identified, 62 were found to be differentially abundant (p<0.05) between men- of Nairobi, Nairobi, strual phases, which included many immune mediators (22). Pathway analysis indicated that remodeling of epithelial adherens junctions pathway (p=6.08 x 10-4) was overabundant during the follicular phase and the Kenya IL-8 signaling pathway (p=4.07 x 10-7) was elevated in the luteal phase. The former pathway is important as it is involved in cell to cell adhesion, which is necessary for maintaining a protective barrier of epithelial cells from the extracellular environment while the latter is involved in immune cell recruitment attracting HIV susceptible cells to the site of infection. Conclusions: This study demonstrates significant differences in immune mediator expression during the follicular phase and luteal phase of the menstrual cycle, which may influence one’s susceptibility to HIV-1 infection. Further studies examining immune cell populations and changes in pro/anti-inflammatory compounds are warranted to further determine if there is a specific window in which women are most vulnerable to HIV infection. A Qualitative Exploration of Factors Affecting Delivery Site (Home, Public or Private Hospitals) Decision-Making Among New Mothers, Pregnant Women, Husbands and Mother-in-Laws in Three Districts in Karnataka, South India Andrea K. Blanchard, Sharon G. Bruce, Lisa Avery

Department of Background: The National Rural Health Mission (NHRM) aims to increase the uptake of safe institutional Community Health delivery among rural communities in Karnataka. The purpose of this study was to explore the factors influencing preference for home, public or private hospital delivery among rural families in the context of the Sciences, Faculty of Karnataka Heath Promotion Trust’s (KHPT) Maternal, Neonatal and Child Health (MNCH) program. Medicine, University of Methods: Local researchers conducted 112 qualitative interviews in 2010 among 30 pregnant women, Manitoba, Winnipeg, 30 mothers of neonates (≤ 30 days), 27 grandmothers and 25 husbands in three north Karnataka districts: Bagalkot, Bellary and Gulbarga. Interviews were conducted in local languages and then translated to MB Kannada and English for analysis. Ethics approval was obtained from St. John’s College, Bangalore and the University of Manitoba Human Research Ethics Board. Results: Geographical and financial access was seen as important barriers in all districts and among those above and below the poverty line. Of greatest concern were high costs at private institutions, continuing fees at public hospitals and the inconsistent receipt of incentives. Thus, many used private facilities for check-ups but deliveries occurred at home or in the public facilities. Yet access issues were ultimately subor- dinate to cultural expectations for comfortable, respectful and safe care. Specifically, the widespread stress placed on high costs at private institutions was fuelled by the perceptions and experiences that public hospitals often provided less quality care than private facilities, particularly in Gulbarga. In addition, while some participants in Bellary and Bagalkot indicated that hospital delivery is becoming more favoured among the younger generation, others did not see the quality as an improvement over home delivery. Conclusions: In the literature, quality of care beyond access has been overlooked in favour of support for incentives on the demand side and more trained doctors, facilities and equipment on the supply side. A more comprehensive approach to quality of care in line with community values and needs is imperative for improving their perceptions and experiences, and ultimately maternal and neonatal health outcomes at the time of delivery. Negative Regulation of PGC-1α by NF-κB Alexandra Blant and Michael Czubryt

Department of The heart has the ability to use both fatty acids (FA) and glucose as an energy source. Under normal condi- Physiology, University tions the adult heart prefers FA, but during ischemia or hypoxia, the preference switches to glucose because less oxygen is required to metabolize it. While beneficial in the short term, long term use of glucose is inef- of Manitoba, Winnipeg, ficient and can lead to an energy deficient state and decompensated hypertrophy. Peroxisome proliferator- MB activated receptor gamma (PPARγ) coactivator-1α (PGC-1α) is a key regulator of the switch from glucose to FA. The NF-κB transcription factor family also plays a key role in the response to injury and stress, acting as a control point from which surrounding microenvironment is appraised. The relationship between PGC-1α and NF-κB in cardiac tissue is already established, but the exact mechanism of their interaction is unclear. The purpose of this work is to shed light on the mechanism by which p65 (the transcriptional activator of the NF-κB complex) represses PGC-1α. In vitro data from our lab has shown that p65 represses PGC-1α promoter activity. Chromatin immunoprecipitation experiments have also shown an increased interaction between PGC-1α and p65 under hypoxic conditions. Furthermore, a mutation in a putative p65 binding site on the PGC-1α promoter renders the PGC-1α promoter unrespon- sive to p65. Mobility shift electrophoresis will be used to confirm that p65 is unable to bind to the mutated form of the PGC-1α promoter. We hypothesize that HDACs may be involved in the repression of the PGC-1α promoter by p65. This will be tested with HDAC and p65 inhibitors. If our hypothesis is correct, an HDAC inhibitor should prevent PGC-1α promoter repression by p65, while a p65 inhibititor should prevent PGC-1α promoter deacetylation. Understanding the molecular biology between PGC-1α and NF-κB pathways is important in understanding the relationship between inflammation and PGC-1α dependent heart dysfunction. The location of the p65 binding site(s) on the PGC-1α promoter, as well as the mode of PGC-1α activation could be useful in future studies which optimize the heart’s response to damage and stress. Examination of MicroRNA-128 Regulation of NMDA Subunits Implied in Prion Infection Amrit S. Boese1,2, Yulian Niu2, Stephanie A. Booth1,2

1Medical Microbiology Introduction: Prion Diseases are a set of neurodegenerative diseases that lead to death; examples include and Infectious Diseases, Creutzfeld-Jakob Disease in humans, mad cow disease and scrapie in sheep. Prion induced neurodegeneration commences long before clinical symptoms appear. Disease is caused by the conversion of normal Faculty of Medicine, cellular prion protein, PrPC, to the misfolded PrPSc, whose accumulation leads to pathology. The purpose University of Manitoba, of our study is to elucidate the relationship between microRNAs (miRs) and neurodegenerative processes 2Molecular that occur throughout the course of disease using a murine model. Previous miR profiling at end stages of scrapie infection in mice revealed a number of disrupted miRs including miR-128. PathoBiology Unit, We are interested in the potential relationship between miR-128 and NMDA related activity that is apparent National Microbiology at preclinical stages of scrapie infection in mice. miR-128 computationally targets a subunit of the NMDA Laboratory, Public receptor, GRIN2D and GRIN2D function is altered in prion null mice. Furthermore, GRIN2B also has an altered expression during preclinical stages of prion infection in mice. Health Agency of Methods: To test the relationship between miR-128 and NMDA receptor subunits, a luciferase reporter Canada, Winnipeg, MB assay was utilized to test miR binding with the 3’UTR of GRIN2A, GRIN2B and GRIN2D respectively. Murine primary hippocampal neurons and primary cortical cells were used to overexpress and knockdown miR- 128 in order to test miR-128’s relationship with GRIN2A/B/D. Glutamate challenges were also performed in primary murine cells to determine if miR-128 altered during glutamate challenge, and if GRIN2A/B/D mRNA and protein levels changed. miR-128 was also knocked down or overexpressed in primary cells, then challenged with glutamate to examine whether miR-128 manipulation could be neurotrophic. Finally, for the in vivo prion disease paradigm, miR-128, GRIN2B and GRIN2D expression was examined in brain sections of a RML Scrapie time course in mice using in situ hybridization and immunohistochemistry. Results/Discussion: Our results indicate there is a relationship between miR-128 and GRIN2D, both in luciferase reporter assays and primary neuron cultures. There are also alterations in expression of miR-128, GRIN2B and GRIN2D throughout the RML Scrapie time course. Further work will yield whether miR-128 is neurotrophic or neurotoxic. Conclusion: miR-128 targets GRIN2D for downregulation. Manitoba Public Health Policy and Childhood Obesity Andrea E. Bombak

Department of Introduction: Approximately a quarter of Manitobans are obese. Extreme obesity is associated with Community Health conditions that may negatively impact individuals’ quality of life and strain the healthcare system. Opti- mizing children’s cardiometabolic health is critical. Individual behaviour and environmental factors have Sciences, University of been implicated in obesity incidence. While environmental reforms are more likely to provide sustainable Manitoba, Winnipeg, population-wide benefits, small-scale initiatives focusing on lifestyle choices are often prioritized in policy MB development. This study’s purpose is to examine the extent to which Manitoba’s public health department emphasizes individual responsibility and/or broader, structural reforms in childhood obesity interventions. Methods: A comprehensive review of the Manitoba Healthy Living, Youth and Seniors website was undertaken in Fall 2011. All on-going and proposed programs that focused on enhancing nutrition, promoting physical activity, or preventing and managing obesity among children and youth were assessed. Programs were categorized based on whether the initiative focused on promoting individual lifestyle changes and/or involved structural, social, or environmental reforms. Results: Manitoba’s anti-obesity programming for children largely involves nodality instruments and some limited treasury instruments. The majority of programming occurs within schools. While this implies an environmental focus, these programs largely deploy the schools to conduct individualistically-focused campaigns to educate and motivate children to adopt healthy behaviours. Some policies, such as the elimination of trans fat in schools, have involved direct provision and a more structural stance. Recent funding pledged to aid in rectifying Northern food insecurity, establishing new school fundraising campaigns, and extending the Children’s Fitness Tax Credit, seems promising in shifting toward more environmentally- focused programs. Conclusions: Manitoba’s government views childhood obesity as a major public health concern and is rhetorically focused on reforming the obesogenic environment and enhancing the convenience and accessibility of healthy living. However only limited environmental reforms have been instituted and focus on encouraging educators and parents to promote children’s healthy choices and create healthy schools and communities. Promisingly, recent progress has been made in school nutrition, tax benefit, and food security policies. Elaboration of Autologous Tissues for Urological Regenerative Medicine Sara Bouhout, Valérie Cattan, Geneviève Bernard, Alexandre Rousseau, Stéphane Chabaud and Stéphane Bolduc

Centre LOEX de Introduction: In North America, 300,000 surgeries per year are needed to treat urinary tract disorders. If l’Université Laval, Génie usual techniques cause significant complications, bioengineered substitutes designed for now raise the problem of biocompatibility. In this context, the feasibility to elaborate autologous urological tissues, free of tissulaire et exogenous matrices and with a well differentiated epithelium (urothelium), was investigated. To achieve this régénération: LOEX du goal, tissues have to be reconstructed in bioreactors that mimic the dynamic of the urinary physiological Centre de recherche environment. The aim of this study was to determine structural and functional characteristics of these new urologic substitutes. FRQS du Centre Methods: Ureters, bladder and urethra were produced within 2 months using the self-assembly approach. hospitalier affilié Here, the mesenchymal cells produced and assembled themselves their own extracellular matrix, on which universitaire de urothelial cells were seeded. Endothelial cells could be also included in the models to form capillary-like structures (CLS). Near in vivo environment cell culture conditions were reached using mechanical stimuli Québec, Département adapted to each physiological features of wanted tissue. Substitute organization was characterized by im- de Chirurgie, Faculté de munolabeling and microscopy, whereas functionality was assessed by mechanical tests and permeability Médecine, Université measurement. Laval, Québec City, QC Results: Urological substitutes display a pluristratified urothelium on uniform matrices. Compared to static configuration, the dynamic culture led to appropriate cell differentiation proved by the presence of cytoker- atin 20 (cell polarity), uroplakins (barrier function) and ZO-1 (tight junctions). Moreover, watertight functions were improved in dynamic cultured substitutes showing comparable profiles to the native tissues. These results coincided with organization of uroplakins into apical plaques observed with electron microscopy. These tissues demonstrated a suitable resistance not only for suturing but also to pressure, and this, even in the presence of CLS throughout the matrix. Conclusion: The substitutes produced by the self-assembly method combined to dynamic culture seems very promising to meet the needs in the urological field: efficient barrier to urea, able to support physiological pressures, free of exogenous biomaterials in order to allow a graft development with its pediatric patients, autologous cells and matrix to reduce inflammation, and finally their potential prevascularization to promote an early inosculation after grafting. Killer Immunoglobulin-like Receptor Genes in Canadian Tuberculosis Patients Kali Braun1, Joyce Wolfe2, Carmen Lopez1, Sandra Kiazyk1,2, T. Blake Ball1,2, Meenu Sharma1,2

1Department of Objective: Although tuberculosis (TB) rates in Canada are declining, incidence in Canadian Aboriginal Medical Microbology, populations and foreign born individuals living in Canada remains high. The objective of this study was to compile killer immunoglobulin-like receptor (KIR) gene profiles found in Canadian born Aboriginals, Cana- University of Manitoba, dian born non-Aboriginals, and foreign born individuals from Manitoba with active, latent, and uninfected 2Public Health Agency TB status. of Canada, Winnipeg, Methods: DNA was extracted from whole blood using the Qiagen DNA Mini kit according to manufacturer’s instructions. KIR profiles were determined in 209 patients (59 active, 46 latent, and 104 uninfected TB con- MB trols; 54 Aboriginal, 77 non-Aboriginal, and 78 foreign born individuals) using the Miltenyi Biotec KIR typing kit. TB disease status was determined by culture and/or clinical diagnosis. Results: Many significant differences were seen in KIR gene frequencies between individuals with and without tuberculosis. KIR2DL2/S2 (P=0.0005/0.0010) and 2DS3 (P=0.0009) were present less often in active TB cases compared to uninfected individuals. In addition, KIR2DSLB was present more often in latent TB cases compared to uninfected individuals (P=0.0125). These trends remain consistent when broken down into Aboriginal, non-Aboriginal and foreign born population groups. Conclusion: This study shows that there are differences in the KIR gene frequency in tuberculosis patients of different population groups. Further investigation is needed to explore the subtleties of these differences using sequencing and/or KIR-HLA association studies. Developmental Delay at 2-3 Years: Relative Importance of Gestational Age and Social Risk Factors Hilary K. Brown1, 2, Kathy Speechley1, 2, 3, Jennifer Macnab1, Renato Natale, 4, M. Karen Campbell1, 2, 3, 4

1University of Western Introduction: The objective of this analysis was to determine the relative importance of late preterm (34-36 Ontario Department weeks) and early term (37-38 weeks) birth and social risk factors (i.e., social context variables and proximal social processes, as conceptualized by Bronfenbrenner) as determinants of developmental delay at 2-3 years. of Epidemiology and Methods: Data were obtained from the National Longitudinal Survey of Children and Youth. Children who Biostatistics, 2Children’s were 0-1 years in Early Child Development Cycles 2-6 were pooled and followed to Cycles 3-7. The study Health Research sample included singletons delivered at 34-41 weeks (N=15,099). Gestational age was determined by maternal report; implausible values (birth weight for gestational age +/- 4 standard deviations from the median) Institute, 3University were excluded. Developmental delay was assessed using the Motor and Social Development Scale. Multi- of Western Ontario variable modified Poisson regression was used to directly estimate adjusted relative risks. Analyses were Department of weighted using normalized longitudinal weights; design variables were controlled for. Results: In the final model, the association between late preterm birth and developmental delay (aRR=1.21, Paediatrics, 4University 95% CI 0.95, 1.53 [p=.12]) and the association between early term birth and developmental delay (aRR=1.10, of Western Ontario 95% CI 0.95, 1.26 [p=.21]) were non-significant. Of the social context variables, family income inadequacy Department of (aRR=1.41, 95% CI 1.08, 1.84) and maternal secondary school education or less (aRR=1.30, 95% CI 1.12, 1.51) Obstetrics & were significantly associated with developmental delay. Of the proximal social processes, negative parenting (aRR=1.35, 95% CI 1.14, 1.62) and inconsistent parenting (aRR=1.32, 95% CI 1.07, 1.63) were also significantly Gynaecology, London associated with developmental delay. ON Conclusion: These results indicate that gestational age was of less importance than social risk factors in determining risk of developmental delay at 2-3 years. Increased surveillance of families with social risk factors may help to identify children in need of early intervention. Challenges measuring gestational age in population surveys are discussed. The PDZ-Binding Domain of the Oncogenic E6 Protein from High Risk Human Papillomaviruses is not Sufficient to Induce the Degradation of the Cancer-Linked MAGUK Proteins Dana Cabiles1,2 and Alberto Severini1,2

1Department of Introduction: High-risk Human papillomavirus (HPV) E6 proteins interact with host cell proteins which Medical Microbiology contain PDZ domains via a C-terminally located PDZ-binding domain. For example, the membrane associated guanylate kinase homologue (MAGUK) family of proteins, which are found at cell junctions. As a result and Infectious Disease, of the interaction with high-risk HPV E6, the MAGUK proteins are degraded. It has been proposed that the E6 University of Manitoba interaction with the MAGUK proteins may contribute to their oncogenicity, as low-risk HPV E6 proteins do 2Public Health Agency not contain a PDZ-binding domain and therefore do not interact with the MAGUK proteins. This study aims to investigate what domains of high-risk HPV E6 proteins are needed in order to achieve MAGUK protein of Canada, Winnipeg, degradation. MB Methods: Wild-type high-risk HPV16E6, low-risk HPV6E6, as well as mutants, were constructed and cloned into vectors which were used in in vitro transcription/translation reactions. The newly synthesized proteins were then incubated with in vitro translated MAGI-1 protein (a member of the MAGUK protein family). MAGI-1 degradation over the course of one hour was observed by western blot using an anti-MAGI-1 antibody. Results: Removal of the PDZ domain from high-risk HPV16E6 abolished MAGI-1 degradation. In contrast, adding the PDZ domain to low-risk HPV6E6 did not allow for MAGI-1 degradation. The ubiquitination domain of HPV16E6 is required for protein degradation, however, when the PDZ domain was added to only this region, MAGI-1 reduction did not occur. Conclusion: The PDZ-binding domain of high risk HPV E6 protein is necessary but not sufficient to induce MAGI-1 degradation. Investigation into the specific domains needed for MAGI-1 degradation is ongoing. Plasma Oxylipin Profiles and the Response to Dietary Modification with Flaxseed Differ as a Function of Age S.P.B. Caligiuri1,2,3, H.M. Aukema1,4 , A. Ravandi1,3,5, G.N. Pierce1,2,3

1Canadian Centre for Introduction: Oxylipins are endogenously produced from polyunsaturated fatty acids and include octa- Agri-food Research in decanoids, eicosanoids, and docosanoids. These metabolites play a key role in preventing or accelerating chronic disease. Despite the physiologic influence of oxylipins, factors such as age and dietary modification Health and Medicine have not been investigated for their influence on the plasma oxylipin profile. (CCARM) and the Methods: Twenty healthy individuals were recruited and grouped into either a younger (18-29 years) or Institute of older (46-69 years) age category and provided muffins containing 30 g of milled flaxseed and were instruct- ed to ingest one muffin/day as a meal replacement for four weeks. Oxylipins were extracted from plasma at Cardiovascular baseline and after four weeks using solid phase extraction and analyzed using HPLC-MS/MS. The method Sciences, 2St. Boniface scanned for 81 oxylipins and 42 were quantified according to the detection/quantification limits. Hospital Research Results: At baseline, the older age group exhibited 23 oxylipins ≥1.5-fold greater than the younger group, including chemotactic hydroxyeicosatetranoic acids, but also potentially protective oxylipins derived from Centre, and the eicosapentanoic acid (EPA) and docosahexanoic acid (DHA). After 4 weeks of dietary flaxseed consumption, 3 Departments of this decreased to 8 oxylipins. Notably, the most chemotactic hydroxyeicosatetranoic acid, 5-HETE, decreased Physiology, 4Human significantly in the older age group to levels equivalent to the younger group after 4 weeks of dietary Nutritional Science, flaxseed consumption (p<0.05). Oxylipins of DHA, 13, 14, and 20-hydroxyDHA, increased in the younger but decreased in the older group after flaxseed consumption (p<0.05). However, the physiological influence of 5 and Internal Medicine, these oxylipins is unexplored. University of Manitoba, Conclusion: Older individuals may have higher levels of both deleterious and protective oxylipins com- Winnipeg, MB pared to their younger counterparts. Dietary modification with functional foods containing flaxseed may be protective as it effectively reduced the highly chemotactic 5-HETE in older participants. Further exploration on longer dietary modification periods and the physiological influence of the novel DHA oxylipins is required. The ENHANCE Exercise Program: Building a Sustainable Exercise and Wellness Program for Head and Neck Cancer Patients Lauren Capozzi, Harold Lau, Barry Bultz, Lisa Shirt, Nicole Culos-Reed

Faculty of Kinesiology, Physical activity (PA) interventions as a method to positively impact symptom management, treatment-re- University of Calgary, lated recovery and quality of life (QoL) have largely excluded head/neck cancer populations. Head/neck cancer patients deal with severe weight loss, with upwards of 70% attributed to lean muscle wasting, leading Tom Baker Cancer to extended recovery times, decreased QoL, and impaired physical functioning. There is a lack of treatment Centre, Calgary, AB options to help sustain or rebuild this wasted muscle, but strength training and PA hold great potential. Additionally, PA may improve adaptation to cancer related stressors into survivorship by enhancing QoL, increasing physical functioning and reducing cancer recurrence. However, only a minority of patients currently meet prescribed PA recommendations, often due to barriers such as lack of information and programming options. Methods: The ENHANCE Program was a structured 12-week group-based exercise program delivered with a collaborative clinical focus. Patients on or off treatment were referred to the program and following assessment and education, engaged in once weekly group exercise classes. Patients were encouraged to include an additional 2-3 days of home-based activity each week. Assessments pre- and post-intervention included physical fitness and functional testing as well as symptom management. Results: Results (n=17) indicate improved fitness in upper and lower body strength and increased aerobic capacity (all p’s<.05). Participants reported a significant decrease in overall tiredness, depression, and drowsi- ness, and improvements in overall well-being (all p’s<.05). They also reported the program helped them to meet their health and wellness goals, manage symptoms, and gain motivation for PA participation. This program facilitated advancements in patient wellness, survivorship, and autonomy, and provided feasibility data for a current RCT. This current study will (1) Examine the impact of timing of a 12-week PA and nutrition intervention (during or following treatment) for HN oncology patients on body composition, recovery, inflammatory response and QoL; and (2) examine outcomes associated with a 12-week follow-up maintenance program, including adherence symptom management and return-to-work. Age and Sex Differences in the Microarchitecture of Human Bone: Implications for Osteoporotic Disease Yasmin Carter1, C. David L. Thomas2, John G. Clement2 and David M.L. Cooper1

1Department of Osteoporosis’s link to age and its preferential bias towards women are well established; surprisingly howev- Anatomy and Cell er, the underlying mechanisms of this disease remain unknown. To further understand and combat this disease, it is imperative that we develop a better understanding of how bone tissue ages and, explore changes Biology, Faculty of in the cellular-level organization of bone with a specific focus on sex differences. Encysted within an inter- Medicine, University of connected network of spaces called lacuna, osteocytes are the most common cell in bone. The purpose of Saskatchewan, this study was to determine whether differences exist in lacunar density, orientation and morphology in the cortex of adults spanning the human lifespan. Anterior blocks from the proximal femoral shaft of 30 females Saskatoon, SK, aged 20-86 and 36 males aged 18-92 were synchrotron radiation micro-CT imaged. The lacunar density 2University of results showed a negative relation with age only for males (p<0.001), although a negative linear trend was Melbourne, Melbourne, observed in females. The hypothesis that decreased bone mass is associated with altered cellular density found no significant relations. The most significant finding was a considerable reduction (~30%) in lacunar Australia volume across the lifespan in female bone (p<0.001) – a pattern not significant in males. Intriguingly, young women exhibited larger lacunae than young men. Associated with this decline, we observed a difference in shape, with older females (50+ years) having flatter and more equant lacunae than younger females (<50 years). The decrease in volume observed in females suggests an overall trend towards smaller lacunae. This appears to be the product of infilling of lacunae with time – a known phenomenon referred to as ‘pericel- lular hypermineralization’– as well as the integration of smaller lacunae during remodeling. Our results suggest that declines in lacunar density, and therefore osteocytes, may not be as important in the degradation of bone quality associated with aging as much as reduction in the volume of the lacunar network. If indeed the fragility associated with senescent bone is due to reduction in cell capacity and not cell death, this presents a potential target for intervention to promote the patency of the network and overall bone health. Thromboxane Receptor Pharmacogenetics and Identification of an Inverse Agonist Raja Chakraborty1, Scott Gleim2, John Hwa2, Shyamala Dakshinamurti3,4, Raj Bhullar1,and Prashen Chelikani1,4

1Oral Biology, The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and University of Manitoba, mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic 2 Internal Medicine, Yale (nsSNP) variants. Interestingly, in TM4 of TP is a novel nsSNP variant, A160T, of unknown structure and func- University School of tion. Here we target Ala160in the TP for detailed structure-function analysis by site-directed mutagenesis. Medicine, New Haven, The mutants A160T and A160V showed a significant decrease in functional receptor expression (Bmax). Molecular modeling analysis revealed that Ala160in the TP plays a critical structural role in packing of TM3 CT, 3Pediatrics and and TM4 helices. The nsSNP variant A160T displayed significant agonist-independent activity (constitu- Physiology, University tive activity). The identification of the first constitutively active and naturally occurring TP nsSNP, gave us a of Manitoba, Winnipeg, unique opportunity to characterize the biochemical pharmacology of TP antagonists. There is a wide range of antagonists available for TP and recent studies show that these antagonists could be beneficial for treat- MB, 4Manitoba Institute ing cardiovascular diseases, platelet disorders, and asthma. Therefore the well know antagonists for TP, SQ, of Child Health Canada, 29548, Ramatroban (Bay U3405), L-670596 and Diclofenac were chosen and studied for their activity on the Winnipeg, MB constitutive activity of the nsSNP variant, A160T. In this way, identification of an inverse agonist for TP may have pharmacotherapeutic relevance within the vascular system. A Bayesian Network Meta-Analysis of Systemic Regimens for Advanced Pancreatic Cancer Kelvin Chan1, Doug Coyle2, Chris Cameron2, Kelly Lien3, Yoo-Joung Ko3

1Division of Background: For advanced pancreatic cancer, many regimens have been compared with gemcitabine (G) in Biostatistics, Dalla Lana randomized control trials (RCTs). Few have been compared with each other directly in RCTs and the relative efficacy and safety among them are unclear. School of Public Methods: A systematic review was performed through MEDLINE, EMBASE, Cochrane Central Register of Health, University of Controlled Trials and ASCO meeting abstracts up to Jan 2013 to identify RCTs that included metastatic Toronto, Toronto, ON, pancreatic cancer comparing the following regimens: G, G+5-flourouracil (GF), G+capecitabine (GCap), G+S1 (GS), G+cisplatin (GCis), G+Oxaliplatin (GOx), G+Erlotinib (GE), G+Abraxane (GA) and FOLFIRINOX. Studies 2University of Ottawa, were reviewed by two authors and discrepancies were resolved by consensus or by a third author. Data Ottawa, ON, including overall survival (OS), progression-free survival (PFS), response rate (RR), and side-effects were ex- 3Sunnybrook Odette tracted. A Bayesian network meta-analysis with random effects was performed using WinBUGS to compare all regimens simultaneously. Cancer Centre, Results: Twenty-one studies involving 5786 patients were identified, with 20 RCTs involving G, 3 with GF, University of Toronto, 4 with GCap, 2 with GS, 7 with GCis, 3 with GOx, 1 with GE, 1 with GA and 1 with FOLFIRINOX. Median OS, Toronto, ON PFS and RR for G arms from all trials were similar, suggesting the absence of significant clinical heterogeneity among RCTs. For OS, the results of the Bayesian network meta-analysis found that the probability that FOLFIRINOX was the best regimen was 71%, while it was 19% for GS, 7% for GA and 2% for GE respectively. The OS hazard ratio (HR) for FOLFIRINOX vs. GS was 0.82 (95% credible region (CR): 0.53-1.35), the OS HR for FOLFIRINOX vs. GA was 0.77 (95% CR: 0.51-1.23), and the OS HR for FOLFIRINOX vs. GE was 0.67 (95% CR: 0.45-1.08). Similar ranking and probabilities were observed for the best regimen for PFS. Conclusions: FOLFIRINOX appeared to be the best regimen for advanced pancreatic cancer probabilistically, with a trend towards improvement in OS and PFS when compared with GS, GA or GE by indirect comparisons. In the absence of direct pairwise comparisons of these regimens from RCTs, network meta-analysis helps synthesize evidence and inform decision making. Orexins are Involved in Expression of Anxiety in a Rat Model of Post-Traumatic Stress Disorder (PTSD) Xiaoyu Chen1, Sa Li1, Huiying Wang1, Lin Zhang2, Maria Vrontakis2, Hugo Bergen2, Gilbert J. Kirouac1,3

1Department of Oral Orexins (hypocretins), which are peptides that are located in neurons of the lateral hypothalamus, have Biology, Faculty of been shown to regulate behavioral arousal. Recent evidence indicates that orexin neurons are activated by stress and play a role in fear and anxiety. Post-traumatic stress disorder (PTSD) is an anxiety disorder that can 2 Dentistry, Department develop when individuals experience a stressful event. Similarly, rats exposed to an acute episode of mod- of Human Anatomy erately intense footshocks develop long lasting fear and anxiety. The present experiment evaluated the ex- and Cell Biology, pression of the mRNA for prepro-orexin peptide (ppOX) in the hypothalamus of rats that were pre-exposed to footshocks (5×2 s episodes of 1.5 mA presented randomly over 3 min). We also examined the effect of an Faculty of Medicine, orexin receptor antagonist (TCS-1102) on shock induced anxiety behavior in the elevated T- maze (ETM) test. 3Department of For these experiments, we took advantage of the fact that shock rats that showed high immobility (High Re- Psychiatry, Faculty of sponders, HR) to a novel tone one day after shock exposure displayed increased anxiety compared to shock rats that showed low immobility (Low responders, LR) and nonshock rats. We measured the levels of ppOX Medicine, University of mRNA at 14 days after shock exposure using real-time reverse transcription polymerase chain reaction. The Manitoba, Winnipeg, results show that the HR expressed more ppOX mRNA compared to the LR and nonshock rats. In another MB group of rats, nonshock, LR, and the HR received TCS-1102 in a dose of 0 mg/kg, 5 mg/kg, or 10 mg/kg i.p. 30 min before being tested on the ETM. The results show that the HR displayed more anxiety (longer latency to explore the open arm) than the LR and nonshock rats. In addition, the 10 mg/kg dose of TCS-1102 decreased the avoidance behavior displayed by the HR suggesting that the drug had an anxiolytic effect specifically on shock rats that show high anxiety levels. This study highlights the importance of individual differences in prepro-orexin mRNA synthesis and anxiety of rats that received footshock stress. These results also support the use of orexin receptor antagonists for the treatment of fear and anxiety to traumatic stress. Identification of Host Cell Interacting Proteins of Hepatitis c Virus Non-Structural 5A Protein by a Novel Tandem Affinity Purification, Mass Spectrometry Based Approach Taeyo Chestley1,2, Sarah Tohme1, Stuart McCorrister1, Garrett Westmacott1, Alberto Severini1,2 and Michael Carpenter1,2

1Division of Viral Hepatitis C virus (HCV) is a hepatotropic pathogen present in approximately 3% of the world’s population Diseases, Public Health with 3-4 million new individuals infected every year. Up to 70% of afflicted individuals progress to a chronic disease associated with the development of hepatic steatosis, cirrhosis and hepatocellular carcinoma. HCV Agency of Canada, encodes 10 major proteins; four structural elements make up the virion while six non-structural proteins are 2Department of principally involved in genome replication and protein processing. A number of the viral proteins have been Medical Microbiology, shown to deregulate host cellular pathways in order to establish an environment more conducive to viral propagation. This ultimately has the potential to contribute to cellular pathogenesis. One viral protein, Non- University of Manitoba, Structural 5A (NS5A) has so far eluded a defined role in HCV replication. Revealing the NS5A-host cell pro- Winnipeg, MB tein interactome is critical to understanding the protein’s function in virus replication and role in pathogenesis. This information may aid in the identification of novel therapeutic targets as NS5A is essential for virus replication. In order to identify cellular interacting proteins, the coding region for HCV NS5A genotype 2 was cloned into a novel tandem affinity tag. The vector contains HIS6 and streptavidin binding coding regions, allowing expression of tagged fusion proteins. Resulting clones were placed into retrovirus transfer vectors and used to transduce HEK 293 cells. Verification of bait protein expression was determined by immunoblot and proper subcellular localization was confirmed by indirect immunofluorescence. Cellular lysates were subjected to two rounds of affinity purification to the NS5A bait protein and captured proteins were identified by mass spectrometry. A candidate list was established by the identification of a protein in NS5A and its absence in control HEK 293 and HEK 293 cells expressing GFP with a minimum of 2 unique peptides and a False Discovery Rate of <1%. Candidate interacting proteins are currently undergoing confirmation by co-immunoprecipitation studies. Additionally, the specific sites of interaction on the viral protein are being determined by using truncated versions of the NS5A bait protein. Phenotypes are also being established using shRNAi knockdowns to each candidate. Analysis of NS5A - host protein interactions will provide further insight into HCV pathogenesis. Endoglin Regulates TGF-b Signaling and Type II Collagen Production in Chondrocytes Y. Chi1 J. Antoniou2 and A. Philip1

1Division of Plastic Transforming growth factor-beta (TGF-β) plays a critical role in the maintenance of articular cartilage. Dereg- Surgery, Department ulation of its activity results in failure of cartilage repair, leading to chronic degenerative joint disorder, such as osteoarthritis (OA). Our research on cartilage repair focuses on the regulation of TGF-β action by endoglin, of Surgery, McGill a TGF-β co-receptor. Our previous work has shown that chondrocytes express endoglin and that it promotes University, 2 Division of TGF-β induced ALK1 (Activin Receptor-Like Kinase 1) pathway while it inhibits ALK5 pathway to decrease Orthopaedic Surgery, type II collagen expression. In the current study, we examined whether endoglin is altered in human OA cartilage and whether it regulates TGF-β signaling and collagen production in vivo using an endoglin deficient Department of Surgery, animal model. McGill University, Cartilage biopsies were taken from adult patients with traumatic open joint injury who have no history Montreal, QC of OA (N) and from OA patients at hip replacement surgery. These biopsies were analyzed by immunohistochemistry (IHC) and Western blot (WB) to compare levels of endoglin expression. For animal studies, we used an endoglin heterozygous mouse (HET, lacking one endoglin allele) since endoglin null mice are embryonically lethal. Our results show that endoglin is overexpressed in OA human cartilage as compared to N cartilage (n=3). Moreover, HET murine chondrocytes showan increased in basal and TGF-β-induced type II collagen levels as compared to WT counterparts (n=5, p <0.05). Analysis of phospho-Smads (1, 2, 3) and total Smads show that their levels are similar in HET and WT chondrocytes suggesting that endoglin may regulate TGF-β signaling through non-Smad pathways in these cells. Altogether, our finding that endoglin expression is upregulated in human OA cartilage suggests that endoglin may play a role in promoting an OA-like phenotype in human chondrocytes. Increased expression of TGF-β-induced type II collagen in endoglin HET mouse chondrocytes suggests that endoglin inhibits TGF-β action and collagen II production in the cartilage in these mice. Determining mechanism by which endoglin regulates TGF-β action in chondrocytes to affect type II collagen production may provide a basis for the development of novel therapeutic approaches to improve cartilage repair. Human Cathelicidin LL-37 and its Derivative Peptide IG-19 Regulate Chronic Inflammatory Cytokine IL-32-Mediated Inflammation and Confer Protection in Inflammatory Arthritis Ka-Yee (Grace) Choi1,2, Leola N.Y. Chow1, Maike Bossert3, Hadeesha Piyadasa1,2, Jude Uzonna2, Thomas Klonisch3, Scott Napper4 and Neeloffer Mookherjee1,2

1Manitoba Centre for Introduction: Human cathelicidin host defence peptide LL-37 contributes to the resolution of infections Proteomics and and pathogen-induced inflammation. This study investigated the effects of LL-37 and its derivatives in ‘sterile’ inflammation. The objective of this study was to define the effects of LL-37 and its derivatives on chronic Systems Biology, inflammatory cytokine IL-32γ-mediated cellular responses. As IL-32 is directly associated with the severity of Department of Internal rheumatoid arthritis (RA), we also evaluated the effect of LL-37-derived synthetic peptide IG-19, in a murine Medicine, 2Department model of RA. Methods: ELISA and flow cytometry were used to monitor cytokine production in human macrophages- of Immunology, and like THP-1 cells and peripheral blood-derived mononuclear cells (PBMC). Immunoprecipitation and Western 3Department of blots were used to evaluate protein phosphorylation. Collagen-induced arthritis (CIA) murine model was Human Anatomy and used to determine the effects of the peptide IG-19 in-vivo. Outcomes in the murine model were analyzed by standardized clinical scores, evaluation of serum levels of cytokines and anti-collagen antibodies, and by Cell Science, University histology. of Manitoba, Winnipeg, Results: We demonstrated that LL-37 and its derivative peptide IG-19 significantly suppressed inflammatory MB, 4Vaccine and cytokine IL-32γ-induced TNFα and IL-1β production, induced anti-inflammatory cytokine IL-1RA, without Infectious Disease neutralizing chemokine responses in human PBMC and macrophages. The Peptides abrogated IL-32γ- induced phosphorylation of Fyn (Y420), which is known to mediate inflammation. The peptides did not Organization, suppress IL-32γ-induced phosphorylation of Akt-1 (T308) and MKP-1 (S359), which are known to negatively University of regulate inflammation. The peptides alone induced the phosphorylation of MKP-1 (S359) and upstream Saskatchewan, kinase p44/42. Administration of the peptide IG-19 significantly decreased disease severity, reduced cellular infiltration into the joints, protected against proteoglycan depletion and cartilage damage, significantly Saskatoon, SK reduced serum levels of TNFα and anti-collagen antibodies, in the CIA mice. Conclusion: Cathelicidin LL-37 and synthetic derivative peptide IG-19 mediate a net anti-inflammatory effect in the presence of the chronic inflammatory cytokine IL-32, by abrogating the Fyn protein tyrosine kinase phosphorylation, and inducing the activity of the negative regulator MKP-1 dual phosphatase. The peptide IG-19 protects the progression of inflammatory arthritis in-vivo. Overall, this study demonstrates the ability of cathelicidin-derived peptides to negatively regulate immune-mediated inflammation. This study provides that rationale for exploring the therapeutic potential of cathelicidin-derived peptides for chronic inflammatory diseases such as RA. The Use of Daily Passive Exercise and the Serotonergic Agonist Quipazine to Restore Monosynaptic Reflex Excitability in Chronic Spinalized Rats J.W. Chopek, C.W. MacDonell, K. Gardiner, P.F. Gardiner

Department of Exercise and pharmacological agents are routinely used separately or in combination to facilitate locomo- Physiology, Spinal Cord tion following a spinal cord injury. However, our understanding of the effect of pharmacological agents such as serotonergic agonists on motor output is limited. Furthermore, the role rehabilitative exercise (i.e. Research Centre, assisted treadmill training, passive cycling) may have on the interaction of such pharmacological agents is University of Manitoba, also unknown. The purpose of our study was to characterize the effect of the serotonergic agonist quipa- Winnipeg, MB zine on flexor and extensor motor output in the chronic spinalized rat preparation. Furthermore, the effect of 3 months of daily passive cycling exercise on the responsiveness of quipazine was also examined. This was achieved by recording the monosynaptic reflex (MSR) of the tibial and peroneal (extensor and flexor, respectively) nerves elicited from stimulation of the L4/L5 dorsal roots pre- and post- quipazine in the adult decerebrate rat subjected to a spinal transection 3 months prior. As well, half of the spinalized rats (n=7) underwent passive cycling exercise for 1 hour daily, 5 days a week for a 3 month period. The passive cycling was achieved by a robotic cycle in which the rat’s hindlimbs were alternatively moved through a full range of motion at 30-50 cycles per minute. It was found that at 3 months following a spinal transection with no exercise (n=6) quipazine had no demonstrable effect on the extensor and flexor MSR respectively, which is different from the large increases seen in the acutely spinalized preparation. However, in rats subjected to daily passive exercise (n=7), quipazine resulted in a significant increase in both the extensor and flexor MSR, with a greater effect on the extensor MSR compared to the flexor MSR (80% vs 17% increase, p≤ 0.05). Our results demonstrate that 1 hour of daily passive exercise for 3 months following spinal transection partially restores the responsiveness of the MSR to quipazine, with the more marked increase in extensor MSR, perhaps reflecting the more pronounced impact of the passive cycling on extensor pathways. The Human Telomerase “Insertion in Fingers Domain” Regulates Enzyme Processivity and Recruitment to Telomeres T.W. Chu2,3, Y. D’Souza1,3, and C. Autexier1,2,3

1Department of Telomerase is composed of a catalytic subunit TERT and a short integral RNA (TR) to template telomere syn- Anatomy and Cell thesis via its unique ability for repeat addition processivity. As a major tumor biomarker, telomerase-specific inhibition should be feasible by targeting domains unique to hTERT, such as the ‘insertion in fingers domain’ 2 Biology, Division of (IFD). Furthermore, previous studies have identified 3 IFD mutations to be associated with various premature Experimental Medicine, aging diseases. Based on the Tribolium castaneum TERT crystal structure, the IFD is found on the periphery Department of of the TERT ring, and is an attractive candidate for the design of specific and accessible telomerase-based therapies. Furthermore, the biochemical, cellular and mechanistic analysis of the premature aging and blood Medicine, McGill disorder-associated mutations in the hTERT IFD will allow a better understanding of the phenotype of these University, 3Bloomfield diseases and uncover potential drug targets and strategies for their treatment. We assessed hTERT variants Centre for Research in with mutations in the ‘Insertion in Fingers’ domain implicated in processivity (V791Y, and five other mutations, including three associated with premature aging syndromes) hTERT-V791Y reconstitutes reduced Aging, Lady Davis levels of DNA synthesis and processivity compared to wild-type (WT) telomerase. hTERT-V791Y- express- Institute, Jewish ing cells harbor short telomeres, measured as signal free ends (SFEs) and undergo apoptosis, likely due to General Hospital, a defect in recruitment of hTERT-V791Y to telomeres. We identified another hTERT variant in the IFD which Montréal, QC displays low levels of enzyme processivity and total DNA synthesis, and cannot confer cellular immortaliza- tion to limited lifespan human cells. This loss-of-function mutant shows a significant decrease in the number of colocalizations with telomeres, suggesting defects in the recruitment to telomeres. We also identified a gain of function hTERT IFD variant with high enzyme processivity and an increased rate of repeat addition compared to WT enzyme. The three hTERT IFD variants associated with premature aging syndromes display a ~50% reduction in enzyme processivity and a slight decrease in the number of associations with telomeres. Moreover, the loss-of-function IFD variants are not defective in assembly with hTR and none behave in a dominant-negative fashion. Our results suggest that recruitment defects can sometimes be masked by activity or processivity defects, underscoring the importance of examining telomere recruitment in telomerase insufficiency diseases. Identifying the Prevalence of Chromosome Instability Within Interval Colorectal Cancer A. Cisyk1,2,3, Z. Lichtensztejn3, R.H. Wightman1,4, H. Singh1,5 and K.J. McManus1,2,3

1University of Introduction: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in Canada. Manitoba, Over 80% of CRCs are sporadic or randomly arising tumors. Recently, it was shown that 1 in 13 individuals diagnosed with CRC in Manitoba had a negative colonoscopy (i.e. no evidence of colorectal neoplasia) in 2 Department of the time period of 6-36 months prior to their diagnosis. This unique subset of tumors, termed Interval CRCs Biochemistry & Medical reduces the effectiveness of all modalities of CRC screening. While there is little information that details the Genetics, 3Manitoba molecular origins of these tumors, one possibility is that they exhibit altered biological properties that may contribute to their rapid growth and proliferation. Chromosome instability, or large changes in chromosome Institute of Cell Biology, numbers, is a phenotype associated with >85% of sporadic CRC and its presence is associated with the 4Department of acquisition of drug resistance, poor prognosis, and aggressive nature of the tumors. We believe that increase Pathology and Grace in chromosome instability is a contributing factor that drives the development of these tumors. Methods and Results: We have established a fluorescentin situ hybridization (FISH) approach to enumer- Hospital and ate three specific chromosomes (8, 11, and 17). Aneuploidy (abnormal chromosome numbers) is often used 5 Department of as a metric for chromosome instability. Any deviation from the expected number of 2 foci/chromosome/ Internal Medicine, nucleus would be indicative of a chromosomal instability phenotype, and this has been confirmed through Winnipeg, MB the utility of diploid and aneuploid cell lines. We have received ethics approval and assembled a Manitoban cohort of Interval and sporadic (control) CRC tumor samples (total 140 samples). Each sample was cored in duplicate to generate 6 mini-tumor arrays. The FISH approach was extrapolated to CRC tissue sections, before evaluating the Manitoban cohort. Applying the approach to the micro-tumor arrays, we have identified a subset of tumors that exhibit chromosome instability. Conclusions: We have developed a FISH approach that can accurately assess chromosome numbers and chromosome instability. We have confirmed our ability to enumerate chromosomes within the micro-arrays and are in the process of analyzing the tumor samples. This work will determine the prevalence and contribution that chromosome instability has in Interval CRCs, and will help improve screening and treatment options. Maternal Depression and School Readiness: A Manitoba Population-Based Study Brenda Comaskey

Department of Setting: Early childhood has been identified as a critical stage of human development that impacts on out- Community Health comes across the lifespan. Exposure to maternal depression during the prenatal, postnatal, toddler, and year prior to school entry periods is associated with poor child outcomes, including school readiness. Factors as- Sciences, University of sociated with maternal depression include family functioning, household demographics and socioeconomic Manitoba, Winnipeg, status (SES). Increased understanding of how these relationships interact and influence childhood develop- MB ment at ages 5-6 years can inform program and policy initiatives. Objectives: The proposed study will 1) investigate the impact maternal depression has on school readiness, as measured by the Early Development Instrument (EDI); 2) identify aspects of maternal depression (timing, chronicity or severity) most predictive of child performance on the EDI; 3) describe patterns of readiness to learn for children whose mothers were depressed; 4) determine if SES and family functioning moderate the relationship between maternal depression and school readiness; and 5) discover whether other factors moderate/reduce the impact of maternal depression on child EDI scores. Methods: Using linked administrative data held at the Manitoba Centre for Health Policy, this longitudinal, population-based study will use a retrospective cohort design with a sample of approximately 18,000 children born in Manitoba between January 1, 2000 and December 31, 2001. Children will be linked to their birth mothers using an anonymous identifier and mothers’ contacts with the health care system for depression (e.g., physician visits, hospitalizations, prescriptions dispensed) will be tracked from one year prior to the child’s birth to approximately six months into the kindergarten when the teacher-completed EDI is administered. This research will explore the relationship between maternal depression and school readiness using Structural Equation Modeling (SEM), which is suitable for complex, multi-level modelling of longitudinal data. Results: Pending Conclusion: This study will identify several aspects of maternal depression on child school readiness. Increased understanding of this relationship has significant clinical, program and policy implications in terms of supporting mothers, children and families in the early childhood period. Results from this study can inform design and delivery of interventions which support positive outcomes at school entry and beyond. Examining Risk Factors for Children in Care in Manitoba Child and Family Services System Yang Cui and Evelyn L. Forget

Department of Setting: Children in care are those who have been deemed in need of protection, such as exposed to abuse, Community Health neglect, maltreatment, disability, death or conflict in the family, require state interventions as determined by provincial legislation. Children in care may have more emotional, social and behavioral problems than chil- Sciences, University of dren in the general population. The main goal of this proposed study would address the gap in knowledge Manitoba, Winnipeg, about the characteristics of both parents and child that predispose a child to out-of-home care. MB Objectives: This study will: 1) identify demographic, socioeconomic, health, educational and geographic characteristics at intake for parents and their children in care; and 2) examine the risk factors related to out- of-home care. Methods: This study is a retrospective cohort study using the administrative data held at Manitoba Centre for Health Policy. The target population will include children aged 0-17 who started out-of-home care in Manitoba urban and rural areas in 2004/05. The estimated sample size is about 2,000. The potential risk factors we will select are based on their availability from the administrative database. They include: age, gender, family income, parents marital status, parents education level, children’s health and educational difficulties, living in Manitoba rural or urban area. The outcome variable is whether or not a child was in out-of-home care. We will run two sets of frequencies to describe the demographic, socioeconomic, health, educational and geographic factors for parents and their children before entering out-of-home care. T-tests will compare means of two sample sets; and chi-square tests will compare proportions of two sample sets. A bivariate analysis will examine the significant association between each factor listed above and children in care. We will then investigate risk factors for out-of-home care using logistic regression modeling. All data manipulation and statistical analyses will be performed using SAS (version 9.3, SAS Institute, Inc., Cary, N.C.). Results: pending Significance: This study will increase our understanding about the risk factors, health and educational needs for the children in care in Manitoba. Our findings will inform policy and practice in children protection services. The Requirement of Microtubule Associated Dynein Proteins for HIV-1 Reverse Transcription and Replication Kallesh Danappa Jayappa, Zhujun Ao, Xiaojian Yao

Laboratory of Introduction: Dynein is a microtubule (MT) associated protein complex that facilitates retrograde transpor- Molecular Human tation of macromolecules. Although Dynein has been implicated in HIV-1 transportation in cytoplasm, the cellular and/or viral factors that mediate HIV-Dynein targeting and HIV-1 cytoplasmic migration is unknown. Retrovirology, In the present study, we have explored the interactions between PIC associated viral proteins and adopter Department of Medical proteins of dynein, and their role in HIV-1 replication. Microbiology, Faculty Methods and Results: By co-immunoprecipitation assay, we have examined the interaction of HIV-1 Integrase (IN), reverse transcriptase (RT), Matrix, and Capsid proteins with Dynein adopter proteins such as of Medicine, University Dynein light chain 1 (DYNLL1), Dynein light chain Tctex-type 1 (Tctex1), and Dynactin p150 in 293 T cells. Re- of Manitoba, Winnipeg, sults showed that only IN was able to interact with DYNLL1. Meanwhile, IN and DYNLL1 were also interacted MB in in vitro, suggesting a direct protein-protein interaction. Also, we have detected the IN/DYNLL1 interaction in HIV-1 infected cells, indicating its functional relevance for replication. Next, to evaluate the functional significance, we used gene knockdown (KD) approach and found that down regulation of DYNLL1 expression has impaired HIV-1 replication at post entry step/s of early stage replication. Significantly, by using real time PCR, we pinpointed the specific impairment of HIV-1 reverse transcription in DYNLL1-KD cells. Our results also revealed that the requirement of DYNLL1 for viral cDNA synthesis is dependent on presence of IN. Since the p21-activated kinase (Pak1) is known to phosphorylate DYNLL1 and modulate its interactions with different cellular proteins, we asked that whether DYNLL1 phosphorylation by Pak1 regulates its interaction with IN and, consequently, modulates HIV-1 reverse transcription. The results showed that while phospho- mimetic DYNLL1S88E mutant displayed a higher level of interaction with IN, treating cells with Pak1inhibitor (IPA-3) has impaired DYNLL1/IN interaction and HIV-1 reverse transcription. Conclusions: Our study provides a first time evidence for DYNLL1/IN interaction and its requirement for HIV-1 reverse transcription. In addition, findings also suggest the involvement of Pak1 and associated signalling pathway for successful recruitment of DYNLL1 to HIV-1 replication complex during infection. Together, it underscores the critical requirement of IN/DYNLL1 interaction for establishment of HIV-1 infection and/or disease progression. Testing for Response shift in Longitudinal Health Related Quality of Life Data Bolanle Dansu1, Lisa Lix1, and Tolulope Sajobi2

1Department of Background: Patient-reported outcomes such as health-related quality of life (HRQOL) are routinely Community Health collected in clinical and epidemiological studies to understand disease progression, treatment efficacy, and burden of disease in chronic disease populations. However, the assessment of longitudinal change in Sciences, University of HRQOL measures can be affected by reprioritization response shift (RS), a change in the relative importance Manitoba, Winnipeg, of the component domains of HRQOL, which can result in biased estimates of change in overall (i.e., summa- MB and 2Department ry) scores. Specifically, when RS is present, conventional statistical methods may underestimate the amount of change in HRQOL outcomes over time. of Community Health Objectives: This study proposes new statistical procedures for testing reprioritization RS in longitudinal Sciences, University of HRQOL data. These procedures are based on the premise that RS occurs when there are significant changes Calgary, Calgary, AB in the relative importance of HRQOL domains over time. Methods: Procedures based on change in discriminant function coefficients of variables over two measurement occasions are proposed to test for reprioritization RS among the HRQOL domains. This includes an asymptotically approximate z-test (Method I) and a theoretically correct t-test (Method II). These procedures are applied to data from the Manitoba Inflammatory Bowel Disease (IBD) Cohort Study. Reprioritization of domains from the disease-specific IBD Questionnaire and the general SF-36 was investigated for cohort members with active and inactive disease symptoms. Results: The analysis is in progress. Based on previous computer simulations that we have undertaken to compare Methods I and Methods II, we expect the procedures to differ in their ability to detect changes in the relative importance of HRQOL domain due to different underlying statistical assumptions and sensitivity to sample size. Conclusion: There is increasing emphasis in the healthcare system on using self-reported outcomes to describe the patient’s perspective on his/her own health. These RS methods will be important analytic tools for clinicians who seek to describe the effect of new treatments on patient outcomes over time. New Evidence of Effects of Health on Labour Force Participation in Canada Pranay Das

Department of Objective: The primary focus of this study is to estimate the impact of health on labor force participation of Economics, University Canadian residents in relation to their country of birth. Methods: Applying logistic regression on Canadian Community Health Survey (CCHS) data, we analyze of Manitoba, Winnipeg, the effects of health conditions on likelihood of residents’ participation in the labor force. We apply single MB equation model as well as simultaneous equation model for participation. The specific hypothesis tested in this analysis is that the health coefficient is different if the person was born in a foreign country, rather he or she was born in Canada. Likewise, the estimated parameters for the interaction terms between place of birth and health is found statistically significant and indicate a differential effect of health on participation for the foreign born and native born. Results: The findings suggest that better health can result in substantially greater labor force participation. The study also demonstrates that the positive effect of health on labor force participation is greater for foreign born residents compared to their native counterparts, and finally. Additionally, the condition of mental health has the largest positive impact on labor force participation for both foreign born and native born residents. Conclusions: Canada is listed as one of the most successful countries that meet the labor market demand for constant growth of good quality labor force. A possible association between immigrants’ health and their participation in the work force has important implications for Canadian immigration policy. Molecular Epidemiology of Extended-Spectrum β-lactamase-, AmpC β-lactamase-, and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae Isolated from Canadian Hospitals: CANWARD 2007 – June 2012 A.J. Denisuik1, P. Lagacé-Wiens1,2, P.J. Simner4, M. Mulvey1,3 M. Baxter1, H.J. Adam 1,2, D.J. Hoban1,2, and G.G. Zhanel1

1University of Objective: To assess the prevalence, patterns of antibiotic resistance, and molecular characteristics of ESBL-, Manitoba, 2Diagnostic AmpC-, and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolated from Canadian hospitals. Services of Manitoba, Methods: 5,842 E. coli and 1,795 K. pneumoniae were collected from January 2007 to June 2012 as part of Winnipeg, MB, the ongoing CANWARD national surveillance study. Antimicrobial susceptibility testing was performed in 3National Microbiology accordance with CLSI guidelines in order to detect putative ESBL-, AmpC-, and carbapenemase-producers. All putative isolates were characterized by PCR and sequencing to detect resistance genes, and by PFGE to Laboratory, Winnipeg, assess clonal spread. The E. coli O25b:H4 ST131 clone was identified by an allele-specific PCR for the pabB MB, 4Mayo Clinic, gene. Rochester, MN Results: The prevalence of ESBL-producing E. coli [2007: 3.4%, 2012 (June): 7.2%], AmpC-producing E. coli [2007: 0.7%, 2012 (June): 2.6%], and ESBL-producing K. pneumoniae [2007: 1.5%, 2012 (June): 2.9%] increased significantly during the study period. Antimicrobials demonstrating the greatest activity against ESBL-producing E. coli, AmpC-producing E. coli, and ESBL-producing K. pneumoniae in this study were colistin, amikacin, ertapenem, and meropenem. Isolates were generally unrelated by PFGE (<80% similarity); however, ST131 was identified among 55.8% and 28.7% (P<0.001) of ESBL-producing E. coli and AmpC- producing E. coli, respectively. CTX-M-15 was the dominant genotype in both ESBL-producing E. coli (66.2%) and ESBL-producing K. pneumoniae (50.0%), while the dominant genotype in AmpC-producing E. coli was CMY-2 (55.7%). Carbapenemase production was identified in 0.03% (n=2) of E. coli and 0.11% (n=2) of K. pneumoniae, with KPC-3 representing the dominant genotype. Conclusions: The prevalence of ESBL- and AmpC-producing E. coli and K. pneumoniae increased significantly between 2007 and 2012, while the prevalence of carbapenemase-producers in Canada remained low (<1%). Compared to AmpC-producing E. coli, ESBL-producing E. coli were significantly associated with multi- drug resistance and the ST131 clone. Understanding Dengue Transmission through Ecohealth Approach in Dhaka, Bangladesh Parnali Dhar Chowdhury1, C. Emdad Haque1, Robbin Lindsay2, Michael A. Drebot2, W. Abdullah Brooks3,4, Kishor Paul3

1University of Introduction: Developing an appropriate multi-disciplinary research design to apply a “Ecohealth Ap- Manitoba, 2National proach” to understand dengue virus (DENV) transmission in Dhaka, Bangladesh, where the 16 million oc- cupants have been exposed to a resurgence of dengue since 2000. Microbiology Methods: To develop a suitable research design, we considered variation in: socio-economic status among Laboratory, Winnipeg, the city-zones, gender inequality, population density, housing, and water supply, waste disposal and sewage MB, 3Johns Hopkins systems. Multiple disciplinary aspects were encapsulated by examination of: i) rates of human exposure to DENV by identifying individuals with IgM and IgG antibodies to DENV and acute cases of illness from hos- University, Baltimore, pitals (47 diagnostic study of suspected hospitalized patients) by identifying the presence of DENV RNA by MD, 4International PCR ; ii) abundance of dengue vector during monsoon and dry seasons in the same households; iii) self-risk Centre for Diarrhoeal perception by the community members; and iv) human organizations responsible for interventions. Data included in the analysis are: a) two vector surveys [i.e., pupal surveys conducted in 847 households (monsoon Disease Research, season 2011) and 459 households (dry season 2012)]; b) two serosurveys [ i.e., serosurveys conducted in Dhaka, Bangladesh 1128 households (pre monsoon season 2012) and 1130 households (630 paired sera during post monsoon season 2012)]; c) socio-demographic survey of 300 households; and d) 12 focus group discussions and eight key informant interviews. Results: Competent dengue vectors were detected in >40% and 12% of households during the monsoon and dry seasons respectively. The monsoon and dry seasonal pupal index were 0.40 and 0.33 respectively for the selected 12 wards. 80% IgG and 17% IgM were positive during pre monsoon serosurvey. Among the IgM positives, in-house PRNTs, using a serial dilution of sera mixed with a DENV serotype, are being carried out. There are significant variations in dengue risk perception between lower and higher socioeconomic groups. Also, experts ranked dengue risk at a much lower level than lay persons and experts emphasized the need for stronger institutional measures to control and prevention. Conclusions: The overall findings of the study will contribute to the advancement of DENV transmission knowledge, and will further the global knowledge of DENV epidemic potential. The Effects of All-Trans Retinoic Acid on Inflammation and Neutrophil Migration to the Lungs during Sendai Virus Infection in Mice Thomas Di Lenardo

Division of For more than eighty years, it was known that vitamin A and its derivatives are capable of directly modu- Experimental Medicine, lating the immune system. This effect is most well known in cases of acute measles virus infection where high dose supplementation with retinol has repeatedly been shown to have a positive effect on disease McGill University, severity. Vitamin A supplementation programs in regions with poor nutrition have also been associated Montreal, QC with a reduction in all-cause mortality in children 5 years of age and younger. Recently it was shown that retinol is capable of inhibiting measles virus replication in vitro in multiple cell lines while all-trans retinoic acid, the active cellular metabolite of retinol, possesses a more potent inhibitory effect. Unfortunately, the in vivo mechanisms behind the beneficial effects of retinoids are not known. This study focuses on Sendai virus infection in BALB/c mice to evaluate the effects of all-trans retinoic acid on this paramyxovirus in vivo. Two subcutaneous injections on day 1 and 3 post-infection of 50ug all- trans retinoic acid per 10g mouse resulted in a significant decrease in neutrophil infiltration to the lung, as well as decreased overall lung pathology by day 5 post-infection. Virus titers in the lungs of infected animals were not affected by retinoid supplementation. Treatment with all-trans retinoic acid was associated with a reduction in the inflammatory cytokines IL-1beta, TNFalpha, IL-6, and IL-3. Similarly, a decrease in Th1 cytokines IL-12 and IFNgamma coincided with an increase in IL-4 production in the lungs. Overall, all-trans retinoic acid reduced inflammation and pathology caused by Sendai virus infection without a direct inhibitory effect on viral replication at the site of infection. Tyrosine Kinase Inhibitor Gefitinib Selectively Induces Apoptosis in ZAP70+ Chronic Lymphocytic Leukemia Cells and Inhibits B Cell Receptor Signaling Rebecca Dielschneider1,2, Wenyan Xiao2, Ju Yoon Yoon1,2, Edward Noh1, James B. Johnston2, Spencer B. Gibson1,2

1Department of Introduction: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, Immunology, Faculty of is characterized by an accumulation of B cells in the peripheral blood, lymph nodes, and bone marrow leading to immunosuppression. Patients can be stratified into two distinct groups based upon the expression Medicine, University of of the tyrosine kinase ZAP70; CLL patients with ≥20% cells staining for the ZAP70 have more aggressive Manitoba, 2CancerCare disease and shorter survival compared with patients with lower ZAP70 expression. Thus, tyrosine kinase in- Manitoba, Winnipeg, hibitors are attractive therapeutic agents. In this study, we investigated the tyrosine kinase inhibitor gefitinib previously shown to inhibit the ZAP70 homolog Syk. Gefitinib is not myelo- or immuno-suppressive as are MB current chemotherapies. Methods: MTT assays were performed to compare gefitinib, erlotinib, and fludarabine 50IC values in primary CLL cells. Viability also tested by staining with Annexin V-FITC and 7AAD. Flow cytometry acquired on BD FACSCalbiur and analyzed using CellQuest Pro. Signaling molecules of drug-treated and anti-IgM-stimulated CLL cells were analyzed by western blot. Raji cells transduced by lentiviral vector. Results: MTT assays showed that gefitinib is effective in primary CLL cells with a preference for ZAP70+ CLL; the median IC50 of gefitinib was 4.5 μM in ZAP70 + patient cells and > 15.0 μM in ZAP70- patient cells. The similar inhibitor erlotinib had no cytotoxicity. Flow cytometry analysis showed a decrease of viable ZAP70+ CLL cells 24 hours post gefitinib treatment. Although gefitinib decreased the viability of the ZAP70+ Jurkat T leukemia cell line, it failed to affect T cells from CLL patients. Gefitinib inhibited both basal and B cell receptor-stimulated tyrosine phosphorylation, and Syk/ZAP70 tyrosine phosphorylation in ZAP70+ CLL cells. Compared with known inhibitors dasatinib and ibrutinib, gefitinib has the same downstream inhibition of Erk and Akt phosphorylation in ZAP70+ cells. The significance of ZAP70 expression was determined by treating the Raji lymphoma B cell line transduced with control or ZAP70 vector; indeed, ZAP70 expression sensitized the B cell line Raji to gefitinib whereas other inhibitors were less effective. Conclusions: Therefore, gefitinib targets the BCR signaling pathway in ZAP70+ CLL cells and holds potential as a targeted therapy for aggressive CLL. Cholesterol Efflux Capacity is Inversely Associated with Severity of Carotid Atherosclerosis and Increases with Time Since Cerebrovascular Event R.J. Doonan, A. Hafiane, J. Gorgui, J. Genest, S.S. Daskalopoulou

Division of Introduction: HDL is thought to exert its atheroprotective role by promoting cholesterol efflux from lipid- Experimental Medicine, laden macrophages. Cholesterol efflux capacity (CEC) has been shown to be inversely associated with carotid intima-media thickness and presence of coronary artery disease. We assessed the hypothesis that CEC McGill University, is associated with severity of carotid erosclerosis and with cerebrovascular symptomatology. Montreal, QC Methods: Symptomatic (n=114) and asymptomatic (n=41) patients with carotid stenosis were recruited from Vascular Surgery at the Royal Victoria and Jewish General hospitals, Montreal, Canada. Carotid Dop- pler ultrasound was performed and stenosis (50-79%, 80-99%) was graded according to velocities. Detailed information on symptomatology obtained. A blood sample was collected on the day of the ultrasound; HDL was obtained by polyethylene glycol precipitation after depletion of apoB-containing lipoproteins. CEC was determined by incubating HDL in cAMP-stimulated J774 mouse peritoneal macrophages for 6 hours. Specific cholesterol efflux was obtained by subtracting total efflux from efflux in non-cAMP stimulated cells. Differences in CEC were assessed using linear regression according to 1) stenosis, 2) symotomatology and, 3) timing of symptomatology. Results: Compared to patients with 50-79% stenosis (n=31), patients with 80-99% stenosis (n=124) had significantly decreased CEC (beta=-2.23, P=0.04) after adjustment for age, sex, apoAI, and systolic BP. CEC was not significantly different between symptomatic or asymptomatic patients. However, in symptomatic patients CEC increased with increasing time since cerebrovascular event. Specifically, compared to 0-30 days (n=72), CEC was non-significantly increased 31-90 days since event (n=31, beta=1.64, P=NS), while increased significantly≥ 90 days since event (n=11, beta=4.48, P=0.01), after adjustment as described above. Conclusion: These results suggest that CEC is inversely associated with severity of carotid stenosis and that CEC increases with increasing time since symptomatic event. This may be related to remodeling of HDL during the acute phase reaction after a recent ischemic event. Universal Anti-Neuraminidase Antibody Inhibiting all Influenza A Subtypes Tracey M. Doyle1,5, Anwar M. Hashem1,3,5, Changgui Li4, Gary Van Domselaar2, Louise Larocque1, Junzhi Wang4, Daryl Smith1,Terry Cyr1, Aaron Farnsworth1, Runtao He2, Aeron C. Hurt7, Earl G. Brown5,6, Xuguang Li1,5,6

1Centre for Vaccine The only universally conserved sequence amongst all influenza A viral neuraminidase (NA) is located be- Evaluation, Health tween amino acids 222-230, but its potential role remains largely unknown. Through an array of experimen- Canada, Ottawa, ON, tal approaches including mutagenesis, reverse genetics and growth kinetics, we found that substitutions 2National Microbiology in this sequence significantly affected viral replication by impairing the catalytic activity, substrate-binding Laboratory, Public Health and thermostability of NA. Universal monoclonal antibody against this peptide provided broad inhibition Agency of Canada, against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses Winnipeg, MB, of mouse-adapted viruses. We further demonstrated that residues within this peptide that are exposed 3 Microbiology on the surface of NA and located in close proximity to the active site, 1222 and E227, are indispensable for Department, Faculty of antibody-mediated inhibition. This unique, highly-conserved linear sequence in viral NA plays critical role in Medicine, King Abdulaziz viral replication and could serve as a potential target in new antiviral and vaccination strategies. University, Jeddah, Saudi Arabia, 4National Institutes for Food and Drug Control, Beijing, China, 5Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 6Emerging Pathogens Research Centre, University of Ottawa, Ottawa, ON, 7WHO Collaborating Centre for Reference and Research on Influenza, Victoria, Australia The Absence of Atypical E2f leads to Ventriculomegaly D. Dugal-Tessier1, D.S. Park 1, G. Leone2 and R.S. Slack1

1Ottawa Health The discovery of neural stem cells within the adult CNS has indicated an enormous potential in facilitating Research Institute - neuronal regeneration after injury. Studies from our laboratory have suggested that manipulation of the Rb/ E2F pathway can directly impact embryonic and adult neurogenesis, thereby having tremendous potential Neuroscience, and for neuronal regeneration therapies. Recently, two new members of the Rb/E2F pathway have been dis- Department of Cellular covered, the atypical E2fs: E2f7 and E2f8. Initial studies have suggested that atypical E2fs regulate diverse and Molecular processes such as cell proliferation, DNA-stress response, apoptosis, however their importance in the brain are unknown. To analyze their function during brain development, we crossed Nestin-Cre mice with mice Medicine, University of bearing a conditional E2f7/E2f8 allele to delete both E2f7 and E2f8 in neural precursor cells. Whereas cortical Ottawa, Ottawa, ON, development was largely unaffected by E2f7/8 deficiency, we observed an enlargement of the lateral ven- 2Human Cancer tricles occurring postnatally, a brain condition named ventriculomegaly. We then looked at ependymal cells which are the cells lining the wall of the lateral ventricles, to determine if these cells were affected by the Genetics Program, absence of atypical E2fs. We found a significant increase of the number of BrdU+ and Ki67+ ependymal cells, Department of suggesting that the absence of E2f7/8 of disrupts their quiescence. In addition, we observed a distortion Molecular Virology, of the ependymal motile cilia and glia cell polarity defect lining of the lateral ventricles, a defect previously Immunology, and linked to ventriculomegaly. In conclusion, our work suggest that E2f7 and E2f8 transcription factors are involved in the integrity and the polarity of ependymal cells, which is crucial with regards to the role of these Medical Genetics, cells in the maintenance of the neural stem cell niche. Comprehensive Cancer Center, Ohio State University, Columbus, OH Mental Health Service Use Patterns by Immigrant Groups and Long Term Residents in Ontario Anna Durbin

Institute of Health Objective: Worldwide Canada has the second largest proportion of foreign born individuals. Annually about Policy, Management one half of immigrants settle in Ontario. Even so, little is known about immigrant use of health services in Ontario. The proposed work will examine access (i.e., any use) and utilization (i.e., number of visits) of health and Evaluation, services for non-psychotic (e.g., depression) and psychotic disorders (e.g., schizophrenia) by immigrants with University of Toronto, different lengths of time in Canada, region of origin, visa type, and area characteristics. This will be compared Toronto, ON to use patterns by long term residents. Methods: Ontario administrative health service use databases will be linked to an immigration database managed by Statistics Canada with information collected from immigrants at arrival to Canada. Data will be accessed at the Institute for Clinical Evaluative Sciences (ICES) in Toronto. The three studies will use different methodologies (cross-sectional, longitudinal, and multi-level) to examine immigrant mental health service use. The predictors of main interest are region of origin (9 groupings); time since immigration; and neighbourhood deprivation. The mental health service use outcomes include use of primary care, psychiatric care, emergency department care, and inpatient admissions. Separate adjusted models will predict use of services for non-psychotic and psychotic disorders. Results: In addition to helping to fill knowledge gaps, the proposed research has methodological advantages over similar existing Canadian research. Specifically, linking immigration and health services databases allows for examining use of varied mental health services by sub-groups of immigrants across time. This marks an advancement over most research that uses Canadian health surveys. These surveys often only include a sample of immigrants who speak English or a small number of other languages; are self-report and therefore are limited by recall, and reporting biases; are usually cross-sectional; and are less complete and accurate than administrative data. Conclusion: The short term knowledge translation goal is to increase decision makers’ awareness of their importance of supporting immigrant mental health. In the longer term, I hope that a more nuanced description of immigrant mental health service use will lead to changes to mental health service delivery and more funding allocated to research on immigrant mental health. Discharge Planning with Older Adults: The Influence of Social, Cultural and Political Systems and Practices Evelyne Durocher

Rehabilitation Sciences, Background: Returning to one’s home environment or moving to a long-term care home after an inpatient University of Toronto, healthcare admission can have tremendous implications for older adults and their families. In the process of assisting patients and families to make discharge decisions, healthcare teams are challenged to balance cli- Toronto, ON ent safety with respect for patients’ autonomous choices, in conjunction with family concerns and priorities. Situations may be further complicated by complex power interrelationships between patients, families and healthcare professionals, and options may be limited by systemic policies and practices. Research purpose: The purpose of this research is twofold: to explore the perspectives of older adults, their involved family members, and the healthcare professionals involved in discharge planning with older adults; and to examine social, political and cultural influences affecting the process, choices, recommendations and decisions of the different individuals involved. Methods: Using ethnographic case study methods, observations and interview data were collected from five case studies, each consisting of an older adult being discharged from inpatient rehabilitation, and their involved family members and healthcare professionals. Taking a critical bioethics perspective and using relational autonomy theory, this research examines how social, cultural and political environments shape discharge planning processes and outcomes for older adults. Results: Medical, social and cultural discourses shape the role expectations and behaviours of individuals involved in the process of discharge planning with older adults and a relational approach rather than a traditional approach to autonomy is applied. Additionally, values embedded in the healthcare system drive discharge processes, which shape outcomes and arguably impede client-centered care for older adults facing discharge from inpatient rehabilitation services. Clinical implications: In complex discharge planning scenarios individuals may be challenged to balance many competing demands and interests, thus complicating and at times lengthening the discharge process, increasing distress for all involved and potentially decreasing both health outcomes for the patient and the efficiency of healthcare systems. This examination of how contextual influences are shaping the process sheds light on the root of some of the challenges that individuals involved in discharge planning are facing, and aims to inform the development of policy to facilitate the provision of ethically supportable discharge planning practices and contribute to more effective and efficient healthcare systems, thus improving the quality of life of older adults. Flaxseed Enterolignans have Antihypertensive Action in PAD Patients in the Flax-PAD Clinical Trial Andrea L. Edel1, Delfin Rodriguez-Leyva2, J. Alejandro Austria1, Wendy Weighell3, Michel Aliani4, Randolph Guzman3 and Grant N. Pierce1

1CCARM, St. Boniface Introduction: Flaxseed, an oilseed largely produced in Canada, is highly prevalent in cardioprotective lig- Hospital, University of nans. Secoisolariciresniol diglucoside (SDG) is the major lignan found in flaxseed whose metabolites, known as enterolignans, demonstrate antioxidant characteristics. Free radical induced damage is thought to be Manitoba, associated with hypertension. Peripheral arterial disease (PAD) is highly related to hypertension. The aim of 2Cardiovascular this study was to determine the effects of milled flaxseed on plasma bioavailability of enterolignans and to Research Division, VI examine the correlation of plasma enterolignans, specifically enterodiol (END), enterolactone (ENL) and total enterolignans (END + ENL) on systolic (SBP) and diastolic (DBP) blood pressure. It was hypothesized that Lenin University PAD patients would exhibit elevated plasma enterolignan concentrations upon consuming flaxseed with an Hospital, Holguin, associated attenuation of blood pressure. Cuba, 3Asper Clinical Methods: A clinical population with PAD was randomized into two groups: a ground flaxseed or whole wheat placebo group in which individuals were required to consume 30g of food varieties daily for 6 Research Institute, months. This single centre, prospective, double-blinded, randomized controlled clinical trial (NCT00781950 4 Human Nutritional at clinicaltrials.gov), known as the Flax-PAD Trial, involved 110 patients >40 years of age with PAD (ABI<0.9). Sciences, University of Plasma enterolignans were measured at 0-, 1- and 6-months using GC-MS. BP was measured using a sphyg- Manitoba, Winnipeg, momanometer. Results: Within the ground flaxseed group, plasma enterolignan levels increased significantly compared to MB baseline and placebo (p = 0.003). Neither body mass index (BMI) nor waist circumference differed between the treatment groups. SBP was reduced by ~10 mm Hg (P < 0.04) and DBP by ~ 7 mm Hg (P < 0.004) in the flaxseed group relative to control following 6 months of intervention. Inverse correlations with DBP were observed with both plasma ENL (r = -0.35; P = 0.02) and Total Enterolignans ( r = -0.4; P = 0.01). Conclusion: PAD patients who consumed 30g of ground flaxseed daily for 6 months had a significant reduction in SBP and DBP. Total plasma enterolignans and plasma ENL concentrations correlated inversely with DBP. Consuming ground flaxseed daily may offer an alternative to blood pressure lowering medications or a more desired naturopathic route. YB-1 Contributes to Sarcoma Metasis via Translational Activation of HIF1α. Amal El-Naggar1,2, Chansey Veinotte3, Cristina Tognon2, Hongwei Cheng4, Syam Somasekharan2, Yuzhuo Wang4, Jason Berman3 and Poul H.B. Sorensen1,2

1Department of Introduction: Sarcomas are a diverse group of malignant neoplasms that are characterized by early meta- Pathology & static spread, and poor prognosis. YB-1 is a member of the highly conserved cold shock domain-containing family of proteins known to bind single and double stranded DNA and single stranded RNA, thereby Laboratory Medicine, controlling transcription and translation of a multitude of genes. Our previous studies using a breast cancer University of British model showed that YB-1 promotes an epithelial-to- mesenchymal transition (EMT) in non-invasive breast Columbia, epithelial cells. In spite of its role in EMT, comprehensive investigations into the role of YB-1 in the development of sarcomas, which are mesenchymal tumors, are currently lacking. 2Department of Methods: To study potential role of YB-1 in human sarcomagenesis, we used MNNG and MG63 (osteosar- Molecular Oncology, coma), TC32 and TC71 (Ewing sarcoma), and Rh30 and Rh18 (rhabdomyosarcoma) cell lines, and performed BC Cancer Research transient and stable YB-1 knockdown (kd) in each cell line. Results: YB-1 kd significantly reduced migration and invasion of each of these cell lines, usingin vitro cell Center, Vancouver, BC, motility and invasion assays. YB-1 kd also profoundly inhibited migration of GFP- labeled human sarcoma 3Department of cell lines xenotransplanted into the yolk sacs zebrafish embryos. Then, using anin vivo renal subcapsule Microbiology and implantation model previously utilized for epithelial tumors, we found that sarcoma cell lines formed highly Immunology, invasive local tumors and metastasized to the lungs within 4-6 weeks. However, local invasion and lung spread were almost completely blocked by YB-1 kd in the same lines. We then assessed potential mecha- Dalhousie University, nisms, and found that YB-1 directly bound and robustly activated the translation of HIF1α mRNAs, while it Halifax, NS, 4Vancouver had no effects on HIF1α transcription. YB-1 itself was robustly induced by hypoxia, and blocking this induc- Prostate Centre, tion blocked HIF1α protein levels under hypoxia. HIF1α kd blocked YB-1 mediated induction of sarcoma cell migration and invasion, and ectopic expression rescued the effects of YB-1 kd on the same parameters. University of British Notably, local implantation site tumors with YB-1 kd demonstrated massive hemorrhage and necrosis com- Columbia, Vancouver, pared to control tumors, and this was significantly correlated with reduced microvessel density and VEGF BC production in the former. Conclusions: These data indicate that YB-1 promotes sarcoma cell metastasis through translational activation of its downstream mediator, HIF1α, and point to potential consequences for sarcoma angiogenesis. Tar- geting YB-1 or its downstream effectors represents a promising future target in the treatment of sarcomas. Quantifying Heart Dose in Left-Sided Breast Cancer Patients after Radiation Therapy using 4D-dose Accumulation Omar El-Sherif, Stewart Gaede

Departments of Introduction: Previous studies have shown an increase in the incidence of late cardiovascular disease in Medical Biophysics and left-sided breast (BREL) cancer patients whom have undergone adjuvant radiation therapy (RT). Hypothesis: This increased risk is correlated to the radiation dose to the heart during RT. Puprpose: To exam- Oncology, Western ine discrepancies in calculated heart dose with/without accounting for respiratory motion during treatment University, London, ON, planning; and to assess if respiratory motion management (specifically deep-inspiration breath-hold (DIBH) Department of Physics and respiratory gating) can be used to further reduce the radiation dose to the heart. Methods: 5 BREL patients underwent the following imaging protocol prior to radiation treatment; free- and Engineering, breathing fast-helical CT (FB-CT), DIBH fast-helical CT, and a 4D-CT. Treatment plans consisted of two wide London Regional parallel opposed tangential beams covering both the left-breast tissue and the internal mammary chain Cancer Program, (IMC) lymph nodes. Treatment plans were initially created on the FB-CT data set. The treatment plans were then superimposed onto the 0% Phase 4D-CT and DIBH CT. Additionally, a rigid probability-weighted 4D– London, ON dose accumulation (4D-DOSE) was performed by superimposing the FB-CT treatment plan onto all phases of the 4D-CT. The probability-weights for each phase were obtained from a respiratory trace obtained from real-time-position management (RPM) system (Varian Medical Systems, Palo Alto, USA) acquired during the 4D-CT scan. Dose-volume histograms for the heart contours were calculated on the FB-CT, DIBH CT, 0% Phase 4D-CT, and 4D-DOSE. Results: mean heart dose across all 5 patients was 594 cGy, 387 cGy, 576 cGy, and 756 cGy for FB-CT, DIBH CT, 0% Phase 4D-CT, and 4D-DOSE respectively. Mean V5Gy across all patients was 19.4%, 15.3%, 19.1, and 21.2% for FB-CT, DIBH CT, 0% Phase 4D-CT, and 4D-DOSE respectively. Mean V25Gy across all patients was 9.0%, 4.2%, 8.6, and 10.2% for FB-CT, DIBH CT, 0% Phase 4D-CT, and 4D-DOSE respectively. Mean V30Gy across all patients was 8.2%, 3.4%, 7.7%, and 9.1% for FB-CT, DIBH CT, 0% Phase 4D-CT, and 4D-DOSE respectively. Conclusions: Accounting for respiratory motion during dose calculations using 4D-DOSE consistently yielded higher heart doses than the traditional FB-CT. Hence, physicians and planners should exercise cau- tion if relying on free-breathing radiotherapy based on standard fast helical CT scans. Diets High in Polyunsaturated Fatty Acids Decrease Fatty Acid Synthase Protein Levels in Adipose Tissue but do not Alter Other Markers of Adipose Function and Inflammation in a Rat Model of Diet-Induced Obesity Jennifer Enns1,2, Danielle Hanke3, Peter Zahradka1,2,3, Carla Taylor1,2,3

1Canadian Centre for Background: Current research suggests that dietary polyunsaturated fatty acids (PUFAs), which include Agri-Food Research in omega-3 and omega-6 fatty acids, may reduce the risk of adverse events in cardiovascular disease. PUFAs may operate, in part, by modulating cell signaling in the adipose tissue, regulating storage and metabolism Health and Medicine, of dietary fats, and in this way they may influence adipocyte function and the development of obesity. The St. Boniface Research purpose of this study was to assess the effect of diets containing high amounts of polyunsaturated fatty Centre, Winnipeg, MB acids (PUFAs) on adipose tissue function. This was done by feeding rats diets containing oils with varying ratios of omega-3:omega-6 fatty acids and polyunsaturated:monounsaturated:saturated fatty acids. 2Department of Methods: Six-week-old Obese Prone Sprague Dawley rats were randomized into six groups and fed high- Physiology, University fat diets (55% energy) for 12 weeks. The fat content of the diets was delivered in the form of different oils: of Manitoba, high-oleic canola, canola, canola-flaxseed blend (3:1), safflower, soybean oil and lard (control). At study end, tissues and serum were collected and stored at -80°C. Epididymal adipose tissue was 3Department of screened by Western blotting for proteins involved in lipid storage and metabolism, adipocyte function, and Human Nutritional inflammation. If significant changes were detected, real-time qPCR was performed to examine gene expres- Sciences, University of sion. Serum was analyzed for markers of inflammation and vascular function. Manitoba, Winnipeg, Findings: A decrease in fatty acid synthase (FAS) protein was observed in adipose tissue of all groups compared to the lard group. Lipid storage markers perilipin and fatty acid binding protein, fatty acid recep- MB tors GPR41, GPR43 and CD36, inflammatory molecules interleukin-18 and monocyte chemoattractant protein (MCP)-1, and adipose function indicators pigment epithelium-derived factor and adiponectin were unchanged. Levels of FAS mRNA were not different among dietary groups. In the serum, levels of leptin, plasminogen activator inhibitor-1 and MCP-1 were also unchanged among groups. Conclusion: The decrease in FAS protein levels observed in adipose tissue of rats fed diets high in PUFAs may be the result of regulation at the post-transcriptional level or translational, independent of the effect of omega-3:omega-6 ratios on other factors related to adipocyte metabolism. Overall, rats fed high-fat diets appear to be largely resistant to changes in dietary fat composition. Temporal and Region Specific Expression of Methyl-CpG Binding Protein Chinelo Ezeonwuka1,2, Carl Olson1,2 and Mojgan Rastegar1,2,3

1Regenerative Methyl-CpG Binding protein 2 (MeCP2) is a nuclear protein that is ubiquitously expressed in mammals and Medicine Program, functions as a transcriptional regulator that binds to methylated DNA. Loss of MeCP2 is considered to be the primary cause of Rett syndrome and several neuropsychiatric disorders. Genetic mouse models with 2 Department of compromised MeCP2 functions demonstrate that homeostatic regulation of MeCP2 is necessary for normal Biochemistry and CNS functioning. The purpose of this study is to investigate the temporal and region specific expression Medical Genetics, of MeCP2 in the in the central nervous system. The characterization of MeCP2 will be determined using techniques such as western blot and quantitative RT-PCR. Current data describes the regional distribution of 3Department of MeCP2E1 in adult murine brain. By studying the expression, we hope to gain insight into functional signifi- Immunology, cance of MeCP2 in the disease phenotypes associated with MeCP2 dysfunction. University of Manitoba, Winnipeg, MB Rapid and Reversible Knockdown of Endogenous Proteins by Peptide- Directed Lysosomal Degradation X. Fan1, W.Y. Jin1, J. Lu1, J. Wang2, Y.T. Wang1,3

1Brain Research Centre Current approaches to alter levels of endogenous proteins via regulating transcription and/or translation and Department of are hindered by many technical difficulties and not clinically feasible. Here we describe a peptide-based method that can rapidly and reversibly knockdown endogenous proteins in intact cells and in vivo. The Medicine, Vancouver peptide consists of the cell membrane penetrating domain, the target protein-binding domain, and the Coastal Health Research signal that directs the peptide-protein complex into lysosomes for degradation. We demonstrated the versa- Institute, University of tility and efficacy of the method to selectively knock downa -synuclein or death associated protein kinase 1 British Columbia, (DAPK1) in HEK cells. We then showed that this peptide approach rapidly and reversibly knocked down en- Vancouver, BC, dogenous DAPK1 in neurons following excitotoxic/free-radical insults, thereby reducing neuronal damages in primary neuronal cultures. Preliminary results in a rat middle cerebral artery occlusion (MCAO) model 2Institute of show that intravenous injection of the DAPK1-targeting peptide can significantly knockdown DAPK1 in the Pharmacology, stroke-afflicted hemisphere, but not in the contralateral hemisphere. This method may represent a versatile Medicine College of and powerful tool not only for deciphering molecular mechanisms in biomedical research laboratories, but Shandong University, also for developing novel therapeutics for certain pathogenic protein-induced diseases in clinical settings. Jinan, People’s Republic of China, 3Translational Medicine Research Center, China Medical University Hospital, and Graduate Institute of Immunology, China Medical University, Taichung, Taiwan Characterization of a Novel Human Nodal Transcript Scott D. Findlay, Lynne-Marie Postovit

Schulich School of Nodal is a member of the transforming growth factor beta (TGF-beta) superfamily that is Medicine and integral to early embryonic development in vertebrates and the maintenance of human embryonic stem (hES) cell pluripotency. Nodal is also often aberrantly expressed in numerous cancers such as breast cancer Dentistry, Western where its expression is positively correlated with disease stage, and promotes tumour growth and metasta- University, London, ON sis in mouse xenograft models. There is currently only one annotated human Nodal isoform. Using qRT-PCR analysis of human pluripotent stem cell lines and a Nodal minigene construct, we demonstrate the alternative splicing of a novel second transcript isoform for human Nodal. Furthermore, we demonstrate that splicing of this isoform is regulated by a rare C/T single nucleotide polymorphism allele at the 5’ splice donor site of an alternatively spliced cassette exon. SNP genotyping data from an International Stem Cell Consortium study of ethnically diverse hES cell lines was interrogated for the SNP allele that promotes alternative Nodal splicing. Intriguingly, this SNP allele is exceedingly rare in male versus female hES cells of European ancestry (1/23 in Males; 11/31 in Females; p=0.008). This suggests the novel Nodal splicing event may be important in controlling hES cell pluripotency, and might help explain the female sex bias observed in hES cells. Owing to a shifted reading frame relative to the annotated Nodal isoform, the predicted protein for the alternatively spliced Nodal transcript has a novel C-terminal sequence. Overexpression studies reveal that the Nodal variant protein is normally secreted from the cell and enzymatically processed to yield a mature peptide. Furthermore, this Nodal variant protein is differentially N-glycosylated relative to annotated Nodal, a feature that has been demonstrated to confer increased stability and signalling range to the Nodal ligand. Ongoing studies are exploring the mechanisms by which the Nodal variant signals, as well as the role of the Nodal variant in the maintenance of hES cell pluripotency and the promotion of breast cancer progression. This work will help further our understanding of Nodal signalling in development and disease. Exogenous Vitamin D Inhibits Human Pro-Inflammatory Cytokine Responses to TLR-4L but not TLR-8L Innate Immune Activation Natascha Fitch1, Allan B. Becker1,2 and Kent T. HayGlass1,2

1Department of Rationale: Population health studies demonstrate that insufficient/deficient Vitamin D status is strongly -as Immunology, sociated with increased upper respiratory tract infections. As low vitamin D status is widespread, it is imperative we understand the role it plays during an immunological response. 2Department of Objectives: Determine the effects of active vitamin D (1,25(OH)2D3) on peripheral blood mononuclear cells Pediatrics and Child (PBMC) cytokine responses elicited by bacterial or viral ligands, and further understand the mechanism by Health, University of which vitamin D regulates innate immune responses. Results: PBMC stimulated with TLR-4L and exogenous 1,25(OH) D exhibited decreased pro-inflammatory Manitoba and 2 3 cytokine production; whereas cultures stimulated with TLR-8L did not exhibit alterations in cytokine pro- Manitoba Institute for duction patterns in the presence of 1,25OHD. Further examination into the vitamin D pathway revealed Child Health, Winnipeg, decreased vitamin D receptor (VDR) expression in TLR-8L stimulated cells than TLR-4L. Interestingly, the vitamin D activating enzyme (CYP27B1) was more abundantly expressed in TLR-8L stimulated cells. MB Conclusions: Data argue that exogenous 1,25OHD has potent immune regulatory effects on cells stimulated with TLR-4L, but not TLR-8L. Our molecular studies show that TLR-8L stimulated cells differ in their expression of VDR and vitamin D related enzymes when compared to TLR-4L stimulated cells. Therefore, future studies will focus on understanding these observed differences between bacterial and viral ligands in response to 1,25OHD. Perspectives on the Long-Term Sustainability of Evidence-Informed Practice Change in Nursing: A Case Study of Key Factors Influencing Changes that Stick versus Changes that Fade Andrea R. Fleiszer, Sonia Semenic, Judith Ritchie, Marie-Claire Richer, Jean-Louis Denis

Ingram School of Background: There is widespread recognition of the troublesome “evaporation effect” that often occurs Nursing, McGill following implementation of costly practice improvement initiatives in healthcare. There are important gaps in theory, research, and practice regarding the concept of sustainability of practice change, the long-term University, Montreal, effects of evidence-informed innovations, and the factors that contribute to sustainability. These gaps are QC particularly pronounced in the field of knowledge translation in nursing. Purpose: This study aimed to explore how organizational and practice changes within a large, multi-site, acute health centre have been sustained or faded over a period of several years following the implementation of an organization-wide patient safety nursing best practice guidelines (BPG) program. The emphasis was on illustrating the constellations of key factors that influence enduring change or decay of change, particularly at the nursing unit level. Methods: A qualitative descriptive case study approach was used to examine enduring practice change among four embedded, contrasting unit subcases, as well as the sustainability of the program at the health centre’s corporate level. Data was collected by means of 39 in-depth key informant interviews, organizational document review, and four formal site visits. Findings: The sustainability of change can be conceptualized according to three core dimensions: maintenance of benefits, routinization / institutionalization, and evolution / ongoing development. Constellations of key factors influencing sustainability / decay are described in terms of interrelationships between: the nature of the innovation itself (e.g., benefits resulting from, relevance of, and nature of BPG-based nursing practice changes); the processes that support the innovation (e.g., activities related to leadership, communication, reminders, performance monitoring, education, collaboration); and the surrounding context (e.g., culture, resources, characteristics of stakeholders). Higher levels of sustainability occur on units: where the BPG practices yield benefits for patients and providers and are relevant to the nature of the patient population and the work of the nurses; where there are cohesive and involved leadership teams who regularly guide team discussion about the BPG practices, provide verbal reminders to staff, engage nurses in individual and team performance monitoring, and respond to changes in circumstances that threaten the sustainability of BPG practices; as well as where unit context is characterized by team collaboration and a sense of account- ability. A Dichotomy of Function: E1A binds hBre1 thereby Blocking Cellular Interferon Transcription and Activating Viral Gene Transcription G.J. Fonseca and J.S. Mymryk

Department of Upon infection, human adenovirus (HAdV) must reprogram the cellular environment for optimal viral rep- Microbiology and lication. This is done via subversion of host cell transcription. Modulation of transcription is orchestrated in large part by the first expressed HAdV gene, Early Region 1A (E1A). E1A binds and appropriates components Immunology, of the cellular transcriptional machinery to alter cellular gene transcription and activate viral early genes University of Western transcription. Here we discuss E1A interaction with cellular hBre1 and the results on both cellular and viral Ontario, London, ON genes. hBre1 is an E3 ubiquitin ligase which functions to monoubiquitinate H2B at lysine 120 (H2B-ub). This histone modification results in drastic increases in transcription. Specifically, we uncovered a role of H2B-ub in transcriptional activation of the cellular type I interferon (IFN) response which is critical for host defense during viral infection. Interestingly, we have found that E1A binds to and inhibits this activity of hBre1 and so blocks H2B-ub. The loss of H2B-ub at interferon genes antagonizes the transcription of these genes, thereby crippling the innate antiviral response. Further, E1A changes the function of hBre1 at viral early genes from a ubiquitin ligase to a scaffold which recruits a known binding partners, hPaf1. hPaf1, a potent transactivator, then induces several histone modifications which are involved in transcriptional activation. Overall, E1A binds to a transactivator, hBre1, at cellular genes and blocks its activity. Then E1A redirects the function of this now defunt transactivator along with hPaf1 at viral genes with the effect of shutting down cellular innate immunity genes while activating other early viral genes. The ability of E1A to target hBre1 to simultaneously repress cellular interferon dependent transcription, while activating viral transcription, represents an elegant example of the incredible economy of action accomplished by a viral regulatory protein through a single protein interaction. Microbial Composition of Drinking Water in Geographically Defined Areas of Low and High Incidence Rates of Inflammatory Bowel Disease in Manitoba, Canada Jessica D. Forbes1,2, Gary Van Domselaar1,2, Charles N. Bernstein3, Denis O. Krause1,4, Ehsan Khafipour1,4

1Department of Inflammatory bowel disease (IBD) represents a group of multifactorial, chronic and debilitating diseases. Medical Microbiology Though the etiology of IBD is largely undefined, it is widely accepted that microbial agents play a critical role in disease etiopathogenesis: through particular commensals or pathogens inducing inflammation and/ and Infectious or dysbiosis of the gut microbiota. Historically, among the highest worldwide incidence and prevalence Diseases, University of rates have been reported in Manitoba, Canada. Geographical areas with low and high incidence rates of IBD Manitoba, 2National (LIA and HIA, respectively) have previously been mapped out across Manitoba. We therefore investigated whether the microbial composition of water could represent a potential source of etiology. Water samples Microbiology were collected from 20 LIA and 20 HIA in Winnipeg, Manitoba and in two rural communities. DNA was Laboratory, Public extracted and V1-V3 regions of bacterial 16S rRNA genes were amplified and sequenced using Roche 454 Health Agency of GS-FLX-Titanium sequencing. Quality control, OTU classification and alpha diversity of sequence reads was performed using the mothur software package. Partial least squares discriminant analysis (PLS-DA) of SIMCA Canada, 3Department was performed on relative abundances of genera to identify those significantly associating with either of Internal Medicine, incidence area. A variety of phyla were represented in the water microbiota; moreover, a plethora of taxa University of Manitoba, were overrepresented and/or underrepresented in either incidence area. PLS-DA identified several genera 4Department of Animal to be significantly associated with either LIA or HIA. Thus, we conclude drinking water in Manitoba provides discrepant microbial ecologies; further, the identification of novel microbes may be instructive in regards to Science, University of disease etiology. Manitoba, Winnipeg, MB Investigating Novel Roles of Cx32 Protein Interactions in Plasma Membrane- Mitochondrial Signaling Complexes Stephanie L. Fowler1,2, Mark Akins1,2, Hu Zhou2,3, Daniel Figeys2, Steffany A.L. Bennett1,2

1Neural Regeneration Introduction: Gap junctions are plasma membrane channels that enable intercellular communication in Laboratory, Ottawa, virtually all cell types of the body. In addition to passing ions and signaling molecules amongst coupled cells, gap junctions act as scaffolds to support transient protein protein interactions that control complex 2 ON, Ottawa Institute intracellular signaling cascades. Very recently it has been confirmed that gap junctions and their substitu- of Systems Biology, ent proteins, the connexins, have important roles in intracellular membranes. An intracellular role has been Department of proposed for the predomoinant liver connexin, Cx32, however, very little is known about the interactome of this connexin, and which intracellular signaling pathways are impacted. Biochemistry, Methods: Here, we used multiple subcellular fractionation techniques to isolate connexin32- enriched Microbiology and membrane microdomains from murine liver. Using a combination of immunoprecipitation-high throughput Immunology, mass spectrometry, reciprocal co-IP, subcellular fractionation methodologies, and confocal microscopy, we report a novel interactome, validated using null-mutant controls. University of Ottawa, Results: Eighteen connexin32 interacting proteins were identified. Surprisingly, the majority of interactions 3 Ottawa, ON, Shanghai were with resident mitochondrial proteins. In particular, Cx32 interacts with connexin26 at the plasma mem- Institute of Materia brane and with the mitochondrial protein, sideroflexin-1. Sideroflexin-1 is thought to transport pyridoxine Medica, Chinese into the mitochondria, which is required for heme synthesis. Cx32-SFXN-1 complex formation increases a small pool of sideroflexin-1 that localizes to the plasma membrane, and Cx32 interaction increases the Academy of Sciences, stability of Cx26 gap junctions. We also show, for the first time, that connexin32 localizes both to the plasma Shanghai, China membrane and the inner mitochondrial membrane of hepatocytes. Conclusion: Bioinformatic analyses support a role for Cx32 in transiently tethering mitochondria to Cx32-enriched membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. With loss of Cx32, the presence of SFXN-1 at the plasma membrane is reduced, and animals exhibit mild hyperferritinemia. These data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome, with links to important mitochondrial functions that may be intervened upon to prevent or treat a variety of hepatic disorders. Regulation of Basal Receptor Tyrosine Kinase Degradation by Ank105 Levi Furber1,2 Dielle Detillieux1 and Deborah Anderson1

1Cancer Research Unit, Overexpression of the 105 kDa isoform of Ankyrin3 (Ank105) promotes basal degradation of pro-oncogenic Saskatchewan Cancer receptor tyrosine kinases (RTK). Activation of Platelet-derived growth factor receptor (PDGFR) activates signaling proteins including phosphatidylinositol 3’ kinase (PI3K)/AKT and mitogen-activated protein kinase Agency and (MAPK) promoting cell proliferation and survival. Deregulation of signaling promotes a tumourigenic pheno- 2Department of type. Expression of Ank105 in NIH 3T3 cells reduces steady state PDGFR levels in a dose-dependent manner, Biochemistry, College thereby attenuating signaling output of MAPK and AKT pathways. Interestingly, Ank105 expression leads to reduction of other RTKs including epidermal growth factor receptor (EGFR). We hypothesize Ank105 reduces of Medicine, University basal PDGFR levels by targeting PDGFR for endocytosis and subsequent lysosomal degradation. In the of Saskatchewan, canonical endocytosis pathway, activated receptors are internalized via clathrin-coated pits in a dynamin de- Saskatoon, SK pendent manner. Internalized vesicles are incorporated into the early endosome where cargo is either sorted for recycling or lysosomal degradation. Treatment of Ank105-expressing cells with a panel of small molecule endocytosis inhibitors demonstrates Ank105 promotes basal PDGFR endocytosis via an alternative, dynamin independent mechanism of internalization. Independence of dynamin activity was confirmed via shRNA knockdown of dynamin-II in Ank105 cells. Additionally, endocytosis inhibitors show that both ligand-induced PDGFR degradation and low density lipoprotein (LDL) uptake proceed by the expected dynamin-dependent mechanism in Ank105 cells. It is the endocytosis of basal, steady state, PDGFR that proceeds by this dynamin- independent mechanism. Lysosomal degradation of internalized receptors required ubiquitination. Ubiquiti- nated receptors are recognized by the endosomal sorting complex required for transport (ESCRT) machinery, incorporated into multivesicular bodies (MVBs) and trafficked to the lysosome in a Rab7-dependent manner. Knockdown of either Tsg101 (an essential ESCRT protein) or Rab7 in Ank105-expressing cells did not recover PDGFR levels; however treatment of cells with lysosomal inhibitors partially recovers PDGFR levels. Interest- ingly, Ank105 interacts with mono-ubiquitin in an in vitro pulldown assay via the spectrin binding domain and co-immunoprecipitates with several ubiquitinated proteins in cells. Treatment of Ank105 cells with proteasomal inhibitors blocks basal but not ligand-induced degradation, suggesting Ank105-mediated basal PDGFR degradation may proceed by an alternative mechanism. Thus, Ank105 lowers steady state levels of pro-oncogenic RTKs by promoting a distinct mechanism of endocytosis and degradation. Time and Dose Dependent Effects of Trans-Fats and Alpha-Linolenic Acid Administration on the Expression of Adiponectin and Markers of Apoptosis in Cardiomyocytes R. Ganguly1,2, T.G. Maddaford1,2, M. Braun1, and G.N. Pierce1,2

1Institute of Introduction: Different types of fatty acids can have very different effects on our cardiovascular health. Elaidic Cardiovascular acid is a trans fat that causes heart disease by inducing atherosclerosis whereas the trans fat vaccenic acid has beneficial cardiovascular effects through its anti-atherogenic properties. Flaxseed contains an omega-3 Sciences and the polyunsaturated fatty acid, α-linolenic acid (ALA) that has also displayed numerous cardioprotective proper- Canadian Centre for ties. In the current study, we will analyze the differential effects of the two trans fats on isolated cardiomyocyte Agri-food Research in viability after 24 and 48 hours of administration. Furthermore, we will investigate the comparative effect of ALA on cardiomyocytes over the same time points. Health and Medicine Results: Elaidic acid was toxic to cardiomyocytes after 48 hour pre-incubation with 400 uM of elaidic acid. (CCARM), St. Boniface TUNEL staining revealed a significant increase in apoptotic cells after 48 hours of incubation. Furthermore, us- Hospital, Winnipeg, MB, ing both immunofluorescence and western blot analysis, elaidic acid treatment of cells for 48 hours increased the expression of the apoptotic marker cleaved caspase-3. Conversely, exposure of cells for 48 hours to ALA in- 2Department of duced significantly less active caspase-3. Vaccenic acid increased caspase-3 and TUNEL staining after 48 hours Physiology, Faculties of but this effect was also significantly less than elaidic acid. Lower concentrations of elaidic acid (50-200 uM) Medicine and induced no changes in caspase-3 after 24 or 48 hours of pre-incubation. Beclin, an autophagic marker, plays a Pharmacy, University of role in cell survival. Western blot analysis revealed less beclin protein present after cell exposure to elaidic acid. There was little change in beclin expression after 24 or 48 hours of exposure to ALA or vaccenic acid. Adipo- Manitoba, Winnipeg, nectin also plays a cardioprotective role within the heart. After 48 hours exposure of cells to elaidic or vaccenic MB acid , there was significantly less adiponectin present in isolated cardiomyocytes as assessed by western blots. Conclusion: High concentrations of elaidic acid directly induced cell death in cardiomyocytes through apoptotic pathways. Elaidic acid also compromised cell survival through an inhibition of autophagy. Both ALA and vaccenic acid are less toxic to cardiomyocytes than elaidic acid. Our data would support the contention that not all trans fats have similar biological effects and the polyunsaturated fatty acid ALA can be cardioprotective. Enhanced ICOS-L Expression on DC in IL-10 Deficiency and its Impact on T Cell Subsets in Respiratory Tract Infection Xiaoling Gao, Jie Yang and Xi Yang

Laboratory for Association of ICOS-L expression and IL-10 production by dendritic cells (DCs) has been commonly found in Infection and infectious disease. The DCs with higher ICOSL expression and IL-10 production are reportedly more efficient in inducing regulatory T cells (Treg). To test the relationship between IL-10 production and ICOS-L expres- Immunity, sion, we examined ICOS-L expression by DC in IL-10 KO mice in Chlamydia muridarum (Cm) lung infection. Departments of Moreover, we examined the development of T cell subsets, including Treg, Th17 and Th1 cell, in the condi- Medical Microbiology tion of IL-10 deficiency and its relationship with ICOSL/ICOS signaling following infection. Surprisingly, we found that the IL-10 KO mice exhibited significantly higher ICOS-L expression by DCs. Moreover, IL-10 KO and Immunology, mice showed lower Treg but higher Th17 and Th1 responses, but only the Th17 response was dependent on Faculty of Medicine, ICOS signaling. Consistently, most of the Th17 cells were ICOS+, while most of the Th1 cells were ICOS- in the University of Manitoba, infected mice. Furthermore, neutralization of IL-17 in IL-10 KO mice significantly exacerbated lung infection. The data suggest that ICOSL expression is independent of IL-10 production and that ICOS-L expression on Winnipeg, MB DC in the presence or absence of IL-10 co-stimulation may respectively promoting Treg or Th17 response, without significant impact on Th1. Assessment of Early Sensory Processing in Infants at High Risk of Autism Spectrum Disorder T. Germani, L. Zwaigenbaum, S. Bryson, J. Brian, W. Roberts, P. Szatmari, C. Roncadin, N. Garon, and T. Vaillancourt

Department of Introduction: Sensory processing is the ability to receive, organize, and interpret sensory stimuli including, Pediatrics, Faculty of but not limited to, tactile, vestibular, and auditory experiences. Difficulties with sensory processing have been reported in children with autism spectrum disorder (ASD). The lnfant(Toddler Sensory Profile (ITSP) Medicine & Dentistry, assesses six subscales (i.e. auditory, visual, tactile, etc.) of sensory processes. No published study has prospec- University of Alberta, tively investigated whether sensory processing in the first 2 years of life is predictive of subsequent ASD Edmonton, AB diagnoses. The objective was to compare ITSP scores at 24 months between 3 groups, based on outcomes at age 3 years: a) high-risk infants (HR; with an older sibling with ASD) subsequently diagnosed with ASD (HR-ASD); b) high-risk infants not diagnosed with ASD (HR-N); and c) low risk infants with no family history of ASD (LR). Methods: Data was collected as part of a longitudinal study aimed at prospectively monitoring HR infants and for comparison, LR infants. The ITSP was administered at 24 months. Assessments at 3 years included the Autism Diagnostic Observation Scale, Autism Diagnostic Interview- Revised, and expert clinical judgment, blind to risk status. A one-way MANOVA was performed to compare groups on overall sensory processing. Groups were also compared on the six sensory processing subscales with pair-wise comparisons, applying a Bonferroni correction for multiple comparisons. Results: There were 34 LR and 63 HR infants (including 15 HR-ASD) who completed the ITSP at a mean age of 24.7±1.1 months. The mean age at the 3-year assessment was 37.6±1.8 months. There was a significant group difference in auditory processing (e.g., startles more easily than others) (F(2,92)=6.87, p=0.0016), with HR-ASD showing more difficulties than HR-N and LR groups. No other group differences were detected. Conclusion: Early auditory processing differences at 24 months were associated with subsequent ASD diagnoses among HR infants and may represent an early risk marker of ASD. Characterization of Emerging Streptococcus pneumoniae Non-vaccine Serotypes 15A, 22F, 33F and 35B in Canada, 2011/12 A. Golden1, H.J. Adam1,2,3, M. Gilmour1,2, M. Baxter1,3, I. Martin4, K.A. Nichol2,3, W. Demczuk4, D.J. Hobani1,2,3, and G.G. Zhanel1,3

1University of Background: The proportion of Streptococcus pneumoniae (SPN) non-vaccine serotypes (STs) circulating in Manitoba, 2Diagnostic Canada rose after the introduction of PCV-7 (2003-04).The SPN Serotyping and Antimicrobial Susceptibil- ity: Assessment for Vaccine Efficacy in Canada (SAVE) study (2011-12) was initiated after the introduction of Services of Manitoba, PCV-13 in Canada (2010). A similar increase in non-vaccine STs is occurring after the introduction of PCV-13; 3Canadian these emerging STs (ESTs) include 15A, 22F, 33F and 35B. The purpose of this study is to assess ST evolution, Antimicrobial antimicrobial susceptibility patterns and changes in virulence of ESTs isolated from SAVE 2011-12. Methods: As part of a collaboration with the NML, the SAVE study collected 2519 SPN isolates in 2011-12 Resistance Alliance from across Canada. Serotyping was performed using the Quellung reaction using commercial antisera (CARA), and 4National (Statens Serum Institute, Copenhagen, Denmark). Antimicrobial susceptibility testing was performed in ac- Microbiology cordance with CLSI methods. EST isolates were characterized for virulence using polymerase chain reaction (PCR) to detect the presence of pili. Laboratory (NML), Results: ESTs accounted for 17% (427/2519) of the total isolates received for SAVE 2011-12 (22F: n=235, Winnipeg, MB 9.7%, 15: n=100, 4.0%, 33F: n=58, 2.4%, 35B: n=34, 1.4%). 53% of ESTs were isolated from males and 48% were isolated from patients ≥65 years of age. The highest proportion of ESTs was found in central Canada (Ontario and Quebec, 18%, 304/1673). 61% (61/100) of 15A isolates were multi-drug resistant (MDR), with resistance to clarithromycin, clindamycin and doxycycline as the most common resistance pattern. 15A isolates were significantly more frequently MDR than vaccine ST 19A (26%, 76/297) isolates (p<0.0001). Pili were found in 21% of 15A and 54% of 35B isolates. In comparison, 66% (230/349) of PCV-13 isolates had pili. Conclusion: In 2011-12, ESTs accounted for 17% of SPN isolates collected in Canada. These STs show significant MDR and virulence due to the presence of pili. SPN STs have constantly evolving genetic and phenotypic properties, necessitating ongoing surveillance to assess the evolution of ESTs. The Macrophage Serves as a Gate-Keeper for the Induction of Protective Host Immunity and Bacterial Dissemination during Chlamydia Infection Eric Gracey, Yuriy Baglaenko, Aifeng Lin, Ali Akram, Robert D. Inman

Department of The obligate intracellular pathogen, Chlamydia, is a common sexually transmitted bacterial infection and a Immunology, leading cause of infectious blindness. Here we demonstrate the central importance of the macrophage in directing a protective immune response to Chlamydia. Intraperitoneal (IP) Chlamydia muridarum infection University of Toronto, resulted in early activation of resident macrophages, as judged by CD80 and MHCI upregulation. To deplete Toronto, ON host macrophages at the site of infection, clodronate-liposomes were injected IP with PBS-liposomes as the control. Subsequent to macrophage depletion, mice were infected IP with Chlamydia and examined at days 2, 7 and 14 post-infection. Significant weight loss was associated with infection of control mice, but unex- pectedly macrophage depletion was protective against this early weight loss. During early infection, robust IFNg production by peritoneal NK and NKT cells was observed using flow cytometry, with macrophages being indispensable for this host innate immune response. During the ontogeny of adaptive immunity, IFNg by CD4 and CD8 T cells also proved to be dependent on an intact macrophage population. Chlamydia growth was controlled in the presence of macrophages, yet in the absence of macrophages, persistent growth was observed using qPCR. Indeed, macrophage depleted mice had greater dissemination of Chlamydia beyond the portal of entry, as reflected by Chlamydia 16sRNA in circulating leukocytes. Adoptive transfers of infected macrophages confirmed that these cells are necessary and sufficient for the IFNg surge by lymphocytes at the site of infection. These data identify the macrophage as the central orchestrator of innate and adaptive immunity to Chla- mydia. These studies suggest that quantitative or qualitative alteration in macrophages may play a key role in the development of Chlamydia-associated diseases. Examining the Molecular Effects of Metformin on Cancer Cell Metabolism Takla Griss

Department of Cancer cells are metabolically unique compared to non-cancerous tissue. One of the main metabolic chang- Physiology, Faculty of es in a cancer cell is enhanced glycolysis for ATP generation and biosynthetic agents, a phenomenon known as the “Warburg Effect”. As such, exploiting the differences in cancer cell metabolism may result in novel Medicine, McGill therapeutic avenues for treatment. Metformin is a widely prescribed drug for type II diabetics. Metformin University, Montreal, acts as an electron transport chain complex I inhibitor. Its action on the liver results in lower systemic blood QC glucose in diabetic patients. Metformin was recently shown to induce non-lethal metabolic modifications in cancer cells. This phenomenon results in metformin behaving as a cytostatic agent. However, the metabolic alterations induced by metformin may allow for synthetic lethality. The metabolic pathways affected by metformin were studied. Metabolic analyses show an increase in glucose uptake, lactate production and ammonia production when cancer cells are treated with metformin over time. GC-MS (with isotopomer labeling) is used to investigate the metabolic pathways that are changed in response to metformin treatment. Initial results show a preference for glycolysis with metformin treatment. Moreover, carbons from heavy-labeled glucose are found mainly in citrate compared to the other metabolites of the TCA cycle. Real- time measurements of mitochondrial and glycolytic activity in cells show that metformin treatment causes a significant decrease in oxygen consumption and combined with an increase in extra cellular acidification rate. Lastly, when comparing the presence and absence of AMPK in metformin treatment, we find that cells lacking AMPK are sensitive to metformin treatment combined with glucose withdrawal as opposed to cells with functional AMPK who are 100% viable under these conditions. These results point toward glutamine metabolism as a means of metabolic adaptation and survival to metformin treatment and will therefore be investigated in the future. We will discuss results from our metabolic analyses in this poster. A Functional Uncoupling of the Trans-Tail Histone Pathway may contribute to Chromosomal Instability Brent J. Guppy and Kirk J. McManus

Manitoba Institute of We hypothesize that altered RNF20 expression, a H2B mono-ubiquitinating enzyme, plays a role in pro- Cell Biology and the moting chromosomal instability. Mono-ubiquitination of histone H2B (H2Bub1) also impacts subsequent methylation events occurring on lysines 4 and 79 of histone H3. Although RNF20 and H2Bub1 have been Department of implicated in human cancers, there is currently no information regarding their spatial and temporal progres- Biochemistry and sion patterns in human cells. In this study, we characterize the spatial and temporal progression patterns of Medical Genetics, RNF20, H2Bub1 and methylated lysine 4 and 79 of histone H3 and propose a role for their involvement in maintaining chromosomal stability. Using indirect immunofluorescence and high-resolution digital mi- University of Manitoba, croscopy we describe the spatial and temporal progression patterns of RNF20, H2Bub1, H3K4 methylations Winnipeg, MB and H3K79 methylations throughout the cell cycle in both transformed and immortalized cell lines. Our data demonstrate that H2Bub1 abundance is greatest in interphase when RNF20 is associated with chromatin and is virtually absent in mitosis when RNF20 is no longer associated with chromatin. We also show that H3K4me2 and H3K79me2 are enriched within the interphase nuclei but that they remain abundant in mitotic chromatin. Most surprisingly, H3K79me2 reaches its maximal abundance during mitosis. We further validated these results using standard Western blot analysis on proteins harvested from synchronized populations. These results suggest there is a functional uncoupling of the trans-tail histone pathway between H2Bub1 and H3K79me2 specifically during mitosis. They further suggest a functional role for H3K79me2 during mitosis, which may contribute to chromosome instability and the development of cancer. Autophagy in Phenoconversion of Differentiated and Undifferentiated Fibroblast Shivika Gupta, Morvarid Kavosh, Shu-Ru Chen, Saeid Ghavami, Sunil G. Rattan, Thomas Klonisch and Ian M.C. Dixon

Department of Background: Cardiac fibrosis is a pathologic condition, where cardiac fibroblast phenoconvert to hyperse- Physiology, Faculty of cretory myofibroblast and formation of fibrotic cardiac tissue. Physiologic autophagy contributes to organ- elle turnover and bioenergetic management of starvation. Autophagy is associated to fibro-proliferative Medicine, University of events. Manitoba, Winnipeg Methods and Results: We investigated the effect of inhibition of autophagy in phenoconversion of 2 MB different types of fibroblasts: 1) Primary (P0) rat cardiac fibroblasts. 2) Mouse embryonic fibroblasts (MEF). Basal autophagy was inhibited in P0 rat fibroblasts [Bafilomycin A1 (Baf-A1) 0-7.5 nM, 3-methyl adenin (3- MA) 0-2.5 mM) while ATG3 and ATG5 knock out model were used in MEF cells. Western blot (WB) analysis, Immunofluorescence (IF), and transmission electron microscopy (TEM) were used to investigate autophagy and fibroblast to myofibroblast phenoconversion. Alpha smooth muscle actinα ( -SMA) and ED-A Fibronec- tin were used as phenoconversion hallmarks while LC3β (LC3-I and LC3-II) were used as autophagometer in all experiments. LC3b punctuate and autophagosome double membrane were investigated in IF and TEM respectively. Significant decrease for myofibroblast markerα ( -SMA, EDA-fibronectin) was observed in P0 fibroblasts treated with Baf-A1 and 3MA (72 hrs) compared to their time match control in paralle to decrease of LC3-II to LC3-I ratio. TEM showed inhibition of autolysosome formation and accumulation of autophagosome in Baf-A1 treated cells. On the contrary MEF’s (ATG3 and ATG5 KO) showed a significant increase of myofibroblast marker α( -SMA and EDA-fibronectin) compared to their correspondence wild type phenotype. IF also confirmed the increase ofα -SMA fiber in ATG3, ATG5 KO MEF cells compared to the correspondence wild type cells while P0 rat treated with Baf-A1 and 3-MA showed a decrease in the same marker compared to the time match control. Conclusion: This study provides a key link between fibroblast phenoconversion and autophagy. However, this autophagic regulation differs in fully differentiated cells (i.e. primary fibroblast) vs. undifferentiated embryonic fibroblast (MEF). This finding shows the differences of autophagy function in cellular phenotype in differentiated and non-differentiated fibroblasts. RNAi Based Identification of Host Cellular Proteases Important for Influenza A Virus Replication Mable W.S. Hagan1,2, Charlene Ranadheera1,2, Anders Leung2, Alex Silaghi1,2, Darwyn Kobasa1,2

1Department of Introduction: Influenza viruses are a serious public health concern in Canada and worldwide. While vac- Medical Microbiology, cines are the most effective option against influenza viruses, it takes at least 8 months to prepare a vaccine for an emergency situation such as a pandemic, leaving antivirals as the frontline treatment until a vaccine University of Manitoba, is available. However, current antivirals that target viral proteins are extremely prone to the development of 2Special Pathogens resistance, limiting their use in large outbreaks and providing a significant priority for the identification of Program, National novel targets for antiviral therapy. Activation of viral hemagglutinin (HA), the receptor binding and fusion protein, which occurs by proteolytic cleavage by cellular proteases, is essential for influenza infection. Rela- Microbiology tively little attention has been given to identifying proteases that activate seasonal human influenza viruses. Laboratory, Winnipeg, Therefore, the goal of this study will be to identify the cellular proteases involved in this crucial HA activation MB step for seasonal and pandemic influenza viruses. Methods: Most cells lines used for the culturing of influenza viruses do not express proteases that can proteolytically activate the viral hemagglutanin (HA) and the in vitro culture of most influenza viruses is dependent on the addition of exogenous trypsin to activate HA. To facilitate the detection of proteases that are important in influenza virus growth, we utilized Caco-2 cells that promote trypsin-independent growth of seasonal (H1N1, H3N2) and pandemic (2009 H1N1, 1918 H1N1) influenza viruses. Furthermore, a seasonal H1N1 GFP expressing influenza virus was generated and used as a tool to screen a human siRNA protease library by measuring GFP expression. Results: Initial screens have led to the identification of several proteases that have already been implicated to play a role in influenza biology, including TMPRSS4, PRSS2 and DPP3. Validation of potential proteases by TCID50 assay and Western blot revealed other novel proteases that may play an important role in influenza replication other than HA activation. Conclusion: The identification of host proteases that are required for influenza replication will expand our knowledge of essential processes in influenza biology and ultimately may provide targets for therapeutic intervention in infection. Insulin Impairs Regulatory T Cell Function: Implications for Obesity Jonathan M. Han, Scott J. Patterson, Jan A. Ehses and Megan K. Levings

Department of Surgery, Chronic inflammation is known to drive metabolic dysregulation in obesity and type 2 diabetes. Although University of British the precise origin of the unchecked inflammatory responses in obesity is unclear, it is known that over-production of pro-inflammatory cytokines such as TNF-a by innate immune cells has a key role in the devel- Columbia, Vancouver, opment of metabolic dysfunction. One key hallmark of obesity is high levels of the pancreatic hormone BC insulin, and we hypothesized that there may be an unknown link between hyperinsulinemia and chronic inflammation. Here we show that high levels of insulin impair the ability of regulatory T cells to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin strongly activates AKT/ mTOR signalling in regulatory T cells, leading to specific inhibition of the production of the anti-inflammatory cytokine IL-10. As a result, insulin hinders the ability of regulatory T cells to suppress the production of TNF-a by macrophages. Regulatory T cells from the visceral adipose tissue of hyperinsulinemic, obese mice also have a decrease in IL-10 production and a parallel increase in production of IFN-g. These data suggest that the hyperinsulinemia associated with obesity may contribute to the development of obesity-associated inflammation via a previously unknown effect on regulatory T cells function. Decreased Resistance Arterial Remodeling and Improved Arterial Compliance in Green Lentil-Fed Spontaneously Hypertensive Rats Matthew Hanson1,4, Peter Zahradka1,2,4, Hope D. Anderson3,4, Carla G. Taylor1,2,4

1Departments of Introduction: Hypertension is a major risk factor for cardiovascular disease, the leading cause of mortality Physiology and worldwide. Increasingly, pharmaceutical treatments are being complimented by dietary changes; recent evidence suggesting that lentils may be beneficial in the treatment of hypertension and its associated 2 Human Nutritional pathologies. This experiment was carried out to determine the effect of lentil-containing diets on hyperten- Sciences, 3Faculty of sion and vascular remodeling in the spontaneously hypertensive rat (SHR), an established model of essential Pharmacy, University of hypertension. Methods: 15-week-old SHR were fed diets containing 30% w/w cooked lentils (mixed, red, or green; lyophi- Manitoba, 4CCARM, St. lized weights used), or pulse-free control diet for 8 weeks. Normotensive WKY were fed either mixed lentil or Boniface Research control diets. Weekly blood pressure (BP) and pulse wave velocity (PWV) measurements were obtained. At Centre, Winnipeg, MB the time of termination, pressure myography was performed on mesenteric arteries. Results: At week 8, the mixed lentil-fed SHR animals had lower diastolic BP and mean arterial pressure than all other SHR, with no changes to systolic BP. There were no changes in PWV as a result of dietary intervention. Control-fed SHR arteries exhibited lower vascular compliance than WKY animals, as indicated by a left- ward shift of the stress-strain relationship. Green lentil-based diet restored arterial compliance a level better than WKY control. With respect to vascular geometry, control-fed SHR arteries exhibited smaller lumen and external diameters, which, in the presence of unchanged media width, resulted in augmented media-lumen ratios. SHR fed red or green lentils had external and lumen diameters that were different than SHR control animals, but not so when compared to WKY control. Conclusion: Green lentils, of all lentil diets, were able to restore vascular compliance to a level greater than normotesive WKY control. All lentil-based diets were able attenuate vascular remodeling present in the SHR. These results indicate that changes in BP, arterial remodeling, and arterial compliance can occur independent of one another. In Vitro and In Vivo Evaluation of Linezolid and Meropenem Against Bacillus anthracis Toxin Production and Spore Formation Breanne Head, Adrienne Meyers and Ethan Rubinstein

Department of Medical Background: Bacillus anthracis is a spore forming, toxin producing bacterium that causes anthrax disease. Microbiology, It is transmitted via contact, ingestion or aerosolization resulting in cutaneous, gastrointestinal or inhalation anthrax respectively. Recently, it has also been shown that anthrax can be caused by the injection of University of Manitoba, contaminated drugs, mainly heroine, in drug addicts. In the case of inhalation anthrax, spores persist within Winnipeg, MB the alveolar macrophage where they can germinate and be disseminated throughout the host causing a systemic infection, meningitis and death. Currently, treatment consists of the administration of doxycycline, penicillin or ciprofloxacin for 60 days however spores can germinate 100 days post infection. Penicillin and tetracycline resistant bioengineered strains have also been described, therefore in a bioterrorist attack it should be assumed that the microorganism used is resistant to first line antibiotics until confirmed otherwise. This study will look at the effect of linezolid and meropenem (alone and in combination) on B. anthracis growth, spore formation and toxin production. It is hypothesized that the combination of bacteriostatic and bactericidal agents will be the most effective at reducing spore and toxin production during infection, compared to treatment with only single antimicrobials. Methods: A hollow fiber model will be used to assess toxin and spore production and vegetative cell growth in vitro. Toxins will be quantified using an ELISA. Spores will be enumerated using the heat shock method and vegetative cells will be quantified by plating serially diluted samples on LB agar. Balb/C mice will be used to look at the efficacy of the drugsin vivo. Blood and lung tissue will be collected at various times post inoculation for further analysis. Spore, toxin and vegetative cells will be quantified as previously mentioned. Relevance: Inhalation anthrax, the most severe form of the disease, is the main focus of this study since it has a mortality rate of 100% if left untreated. Since B. anthracis is easily disseminated and can cause high mortality and morbidity it is a great concern as a bioterrorism weapon therefore better therapeutics are of high priority. Changes in Axon Diameter Distributions as a Potential Biomarker for Alzheimer’s Disease Sheryl L. Herrera1, Trevor J. Vincent2, Jonathan D. Thiessen1,3, Richard Buist3, and Melanie Martin1,2,3

1Physics and Alzheimer’s disease (AD) is putting an unprecedented strain on Canada’s ailing health system. AD and other Astronomy, University forms of dementia affect almost 20,000 Manitobans, a number that is expected to rise to more than 34,000 by 2038. The total economic burden of dementia in Manitoba is 885 million dollars and is expected to rise to of Manitoba, 4.4 billion by 2038. It is estimated that 500,000 Canadians are affected by AD with the number expecting to 2Radiology, University double within a generation. While there is an increase in the understanding of this disease, there lacks a sen- of Manitoba, Winnipeg, sitive and objective in vivo central nervous system (CNS) biomarker which could help translate laboratory findings. A biomarker would play an important role in the evaluation of novel therapeutics and could thus MB, 3Physics, University lead to faster development of effective treatments. A biomarker would provide insight into the biological of Winnipeg, Winnipeg, and anatomical factors which lead to the complex clinical features of AD. MB Using diffusion MRI, I am developing and optimizing a new method for the measurement of the size of very small (1 µm) tissue structures, such as axon diameters. Measurements can be taken as a function of diffusion time, providing information about the structure in which the molecules are diffusing. The new method will have important biological and neuroscience applications; one example is probing axon diameter distributions or fibre composition. Current state-of-the-art methods cannot distinguish these small structures as the pulse sequences that are used limit the ability to probe the shortest diffusion times. To circumvent those limitations, this new method will use oscillating gradients in the place of pulsed gradients, thus relying on probing the shortest possible diffusion time scales to enable the transition from restricted to free diffusion within the smallest structures. Computer simulation results from this pulse sequence show our ability to distinguish cellular sizes as small as 1 µm. Sizes on the order of 5-10 µm are easily distinguished with lower frequencies than smaller sizes of 1-3 µm. MRIs of celery phantoms indicate the feasibility of running the method on our 7 T 21 cm bore MR imager. Winnipeg’s Homeless in the At Home/Chez Soi Multi-Site Trial Aynslie Hinds3, Jino Distastio1, Patricia Martens2,3, Mark Smith2,3

1Institute of Urban Objectives: Homeless individuals with poor health frequently use health care services. Many studies on Studies, University of health and health care use among the homeless rely on self-reports, which may be subject to biases. The objectives of this study were to describe the health status and health care use among mentally ill homeless Winnipeg, Winnipeg, individuals, and to compare self-reports and administrative data claims to estimate the degree of agree- MB, 2Manitoba Centre ment between these two data sources. for Health Policy, Methods: Baseline survey data from 100 participants of the Winnipeg site of the Mental Health Commission of Canada’s At Home /Chez Soi research project were linked to de-identified administrative health records Winnipeg, MB, stored in the Repository at the Manitoba Centre for Health Policy. Demographic characteristics and health 3Department of service use were analyzed, as well as disease status for asthma, hypertension, arthritis, and diabetes (us- Community Health ing previously validated definitions). Participants were similarly classified using their survey responses. The degree of agreement between the two data sources was evaluated using cross tabulations and the Kappa Sciences, University of statistic. Manitoba, Winnipeg, Results: There was 100% linkage of surveyed homeless people with the Repository data. In one year, 97% MB of participants had at least one ambulatory physician visit, with an age- and sex-adjusted rate of 14.82 per person-year (Manitoba rate = 4.99 per person-year). 34% had an inpatient hospitalization (adjusted hospital separation rate = 491 per thousand person-years versus the Manitoba rate of 137 per thousand person- years). The degree of agreement between the data sources ranged from a Kappa of 0.27 for arthritis to 0.57 for hypertension. Individuals were more likely to be classified as having one of the four conditions based on the administrative data than on the survey data. Conclusions: Compared to the general population, participants had high health service use. There was poor to moderate agreement between the survey and administrative data. These data sources have different strengths and weaknesses. Researchers studying homeless persons with mental illness should consider using multiple data sources to estimate disease prevalence and health service use. Hepatitis B Virus Activates NF-κB Signal Transduction by Interacting with AIB-1 oncoprotein Andy Hong1, Dong Dong Han4, Christine J. Wright1, Tanya Burch1, Jessica Piper1, Carla Osiowy1,2 Chunjin Gao4, Shirley Chiang1, Travis Magill1, Kevin Dick1, Timothy F. Booth1,2 Xuguang Li3, and Runtao He1,2

1National Microbiology Hepatitis B virus (HBV) is a major global health concern. More than 300 million people worldwide have Laboratory, Public chronic HBV infection, and 15-40% of these carriers will develop liver disease, including hepatocellular carcinoma (HCC), liver failure, and cirrhosis. HBV X antigen (HBx) is a 17 kDa transcriptional trans-activator essen- Health Agency of tial for viral gene expression and replication. HBx was shown to be involved in tumorigenesis by interacting Canada, Winnipeg, MB, with various host cellular proteins, such as AP1, CREB, and NF-κB, involved in signal transduction. Interest- 2Department of ingly, these factors interact with the steroid receptor coactivators (SRCs), a member of which is the AIB-1 (amplified in breast cancer 1) that has been implicated in several cancers, including breast, prostate, and Medical Microbiology, colorectal cancer. In this study, we report a strong interaction between HBx and AIB-1. A serine/proline motif University of Manitoba, (SSPSPS) in HBx and two LXD motifs (LLRNSL and LLDQLHTLL) in AIB-1 were required for their interaction. Winnipeg, MB, 3Center HBx mutant with a single amino acid substitution that could not bind to AIB-1 had a dramatically reduced ability to activate NF- κB pathway than the wild-type. This suggests an important ant role of AIB-1 in mediat- for Vaccine Evaluation, ing HBx function through direct protein-protein interaction. The findings of this study contribute to the new Health Canada, Ottawa, understanding on the mechanisms with which host signal transduction pathway is regulated by HBx. ON, and 4Beijing Chao Yang Hospital, Affiliated Hospital of Capital Medical University, Beijing, China Self-Regulation, Executive Function & Childhood Obesity: A Theoretical Model Sarah Hutchison and Ulrich Müller

Department of Emerging research suggests that deficits in Execution Function (EF) have been associated with obesity (e.g. Psychology, University Lokken et al., 2009). EF broadly refers to cognitive processes that are involved in the conscious control of action and thought and includes working memory, inhibitory control, and shifting, among other processes. of Victoria, Victoria, BC EF overlaps with another theoretical construct, self-regulation (SR), which refers to: a) commitment to standards, b) monitoring of the self and it's behaviors, and c) the capacity to make change. EF can be conceptualized as a process mediator to SR (Hofmann et al., 2012). EF's (working memory, inhibitory control, and shifting) support mechanisms in an individual's ability to develop commitments according to standards, to monitor the self, and to make changes. While there is some overlap between EF and SR, obesity literature outlines these two theoretical constructs separately. This theoretical model tests the relation between EF, SR and obesity in children (age 8-12). Based on previous research the hypotheses are: 1) Obese children will perform less well on tasks measuring inhibition, shifting, and SR, 2) EF will mediate the relation between self- regulation and obesity status. Mediation can be confirmed if EF and SR measures are separately significantly correlated with obesity status, and if the relationship between SR and obesity status diminishes when EF measures are in the model. This poster will also include implications of the model. Molecular Mechanism of R181Q and R274Q, two VANGL Variants Associated with Neural Tube Defects in Humans Alexandra Iliescu, Michel Gravel, Cynthia Horth and Philippe Gros

Department of Neural tube defects (NTDs) such as spina bifida and anencephaly constitute one of the most common con- Biochemistry and genital anomalies in humans, affecting 1:1000 live births. Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis), which is caused by mutations in the Vangl2 gene (D255E, R263L, S464N) Complex Traits Vangl1 and Vangl2 are membrane proteins that play critical roles in development such as the establishment Program, McGill of planar cell polarity (PCP) in epithelial layers and convergent extension movements during neurogenesis. University, Montreal, QC To date, mutations identified in both Vangl1 and Vangl2 have been associated with neural tube defects in mice and humans. The biochemical characterization of Lp mutants has provided an experimental framework to study the molecular basis of loss-of-function of patient- specific VANGL1/VANGL2 variants detected in co- horts of human cases of NTDs. Of particular interest are invariant residues R181 and R274. R181 is independently mutated to H (VANGL2R177H) and Q (VANGL1R181Q) in two patients, and R274 is mutated to H (VAN- GL2R270H) and Q (VANGL1R274Q) in two other patients, suggesting a critical role for these two variants. Here, we report a common biochemical mechanism for the Lp and for these two human variants. Experiments in stable transfected MDCK cells show that while WT Vangl protein is targeted to the plasma membrane, mutant variants are retained intracellularly in the endoplasmic reticulum, co-localising with ER chaperone calreticulin. In addition to decreased targeting to their site of biological function, mutant proteins are also more rapidly degraded in a cyclohexamide sensitive fashion, have reduced stability and a diminished half- life. Our study highlights a critical role for these mutations on normal folding and processing of Vangl proteins, with highly conservative substitutions not tolerated at these sites. miR-17-92 as a Metabolic Regulator in Cancer Said Izreig, Russell G. Jones

Goodman Cancer Rationale: Cancer is a genetic disease characterized by unrestrained proliferation. Regulators of gene Research Centre, expression are often found dysregulated in cancer. Recently, the role of micro-RNAs (miRNA) in cancer has become a focus of study. miRNAs are small, non- coding RNAs that negatively regulate the translation of their Department of mRNA targets. The polycistronic, oncogenic miR-17-92 cluster is markedly upregulated in many cancer types, Physiology, McGill including hematopoietic malignancies. miR-17-92 targets numerous tumour suppressors. Some of these tu- University, Montreal, mour suppressors, including PRKAA1 (AMPK α1) and PTEN, act as regulators of cell metabolism. Because the fueling of unrestrained proliferation of cancer cells requires a reconfiguration of metabolic activity, regulators QC of cell metabolism warrant study. Despite the established role of miR-17-92 in promoting cancer development and progression, nothing is known of its role in regulating cancer cell metabolism. My project aims to define the role of miR-17-92 in regulating cell metabolism in cancer. Methods: B-cell lymphoma cells with constitutive Myc expression and conditionally deleted miR-17-92 were used. Stable cell lines overexpressing different components of the miR-17-92 cluster were generated from the parental and miR-17-92 deleted B-cell lymphoma lines and mouse embryonic fibroblasts. Extracellular acidification and oxygen consumption rates were measured using the Seahorse extracellular flux analyzer. Glucose and glutamine consumption, and lactate production were measured using the NOVA bioanalyzer with cultured media samples. Isotopomer labeling and GC-MS analysis was used to track metabolic flux through glycolysis and the TCA cycle. Results: miR-17-92 deletion in B-cell lymphoma cells produced a significant drop in lactate production, extracellular acidification, and glucose and glutamine consumption with no measurable impact on oxygen consumption. Conversely, miR-17-92 overexpression was found to enhance glycolytic metabolism, which is reminiscent of highly aggressive cancer cells. Metabolic flux analysis suggested that miR-17-92 overexpression promotes increases in total abundance of glycolytic and TCA cycle intermediate metabolites. Conclusion: These data suggest that miR-17-92 is able to exert a pro-glycolytic influence on cellular metabolism. Importantly, this profile of metabolism is characteristic of some aggressive cancers, suggesting that the metabolic effects exerted by miR-17-92 may contribute to its oncogenic behavior. Further study will examine the feasibility of targeting miR-17-92 to affect metabolic activity in cancer. Role of Non Smad Signaling Pathways and Secretory Leukocyte Protease Inhibitor in Regulating TGF Beta Induced Expression of Neurocan Naima Jahan, Sari S. Hannila

Department of Human Axonal regeneration after spinal cord injury is inhibited by chondroitin sulphate proteoglycans (CSPGs) Anatomy and Cell such as neurocan, which are expressed in the glial scar. Neurocan expression can be induced by transforming growth factor (TGF), which suggests that inhibiting TGF signaling may reduce neurocan levels following Science, University of injury. A recent study from our laboratory showed that elevation of cyclic AMP (cAMP) could reduce levels Manitoba, Winnipeg, of the TGF signaling protein Smad2 in neurons, and we have now found that cAMP significantly reduces MB Smad2 levels in astrocytes. However, when cAMP-treated astrocytes were exposed to TGF, neurocan expression was still strongly induced, which suggests that TGF may mediate neurocan expression through non- Smad pathways. To test this hypothesis, astrocytes were first incubated with either Smad2 or Smad4 siRNA, and then treated with TGF. TGF-induced neurocan expression was still observed in the absence of Smad2 or Smad4, indicating that Smad signaling is not required for neurocan expression. When astrocytes were treated with inhibitors of either the PI3K-Akt or Erk pathways, we observed significant reductions in neurocan, which suggests that these pathways are responsible for mediating neurocan expression. In addition, we have found that astrocytes from mice lacking expression of secretory leukocyte protease inhibitor (SLPI) have significantly lower levels of neurocan compared to wild type astrocytes. Targeting non-Smad signaling pathways and SLPI may therefore be effective strategies to reduce neurocan expression following spinal cord injury. Interaction of TAPP Adapter Proteins with Phosphatidylinositol(3,4)-bisphosphate Regulates Germinal Centre Response and Development of Autoimmunity Nipun Jayachandran, Ivan Landego and Aaron J. Marshall

Department of Introduction: Activation of PI3-kinase enzymes is essential for lymphocyte development and function. Immunology, Active PI3Ks generate several types of D3 phosphoinositides, including PI(3,4,5)P3 and PI(3,4)P2. Tandem PH domain containing proteins (TAPPs) are adaptor molecules which bind specifically to PI(3,4)P2 via their University of Manitoba, C terminal PH domain. This interaction leads to translocation of TAPPs to the plasma membrane. However, Winnipeg, MB their functions in immune cells are largely unknown. Materials: We have performed studies on TAPP mutant mice, in which a mutation was introduced to the C terminal domain of both TAPP1 and TAPP2, to determine the effect of uncoupling TAPPs from PI(3,4)P2. Results: We found that these TAPP KI mice exhibit elevated serum antibody levels and also develop autoan- tibodies and kidney pathology resembling that of lupus. TAPP KI mice generated elevated germinal center (GC) responses upon immunization with T-dependent antigen, suggesting an inhibitory role associated with TAPPs in lymphocytes. The GC B cell population was increased in frequency and showed reduced expression of apoptosis markers, and an expansion of the follicular helper T cell subset was found. We hypothesize that TAPP KI mice have disruption in a regulatory mechanism within the GC leading to sustained B cell activation and survival, and contributing to development of autoimmunity. TAPP KI B cells were found to be hyper-re- sponsive to BCR cross-linking in vitro, and T cell responses are currently being assessed. Our goal is to define the B cell and T cell intrinsic signaling mechanisms enhanced as a result of disrupted inhibitory signaling mediated by TAPPs. Simvastatin Significantly Augments Impact of Fluticasone on Allergic Airway Inflammation and Hyperreactivity in Mice Aruni Jha, Sujata Basu, Min Hyung Ryu, Oluwaseun Ojo and Andrew J. Halayko

Departments of Introduction: Asthma affects over 200 million people worldwide. The disease is refractory to first line Physiology and therapy in about 10% of patients, posing a significant economic and health care burden, which mandates improved or alternate therapeutic strategies. Inhaled corticosteroids are the mainstay of asthma control- Internal Medicine, ler therapy. Statins have emerged as compounds with pleiotropic effects, and their use is linked to reduced University of Manitoba, inflammation and improved lung health. Acute statin therapy suppresses allergic airway inflammation and and Biology of hyperresponsiveness in mice, but impact in humans is equivocal. We hypothesized that combined use of simvastatin and fluticasone enhances the impact of either drug alone in a murine model of allergic airway Breathing Group, inflammation. Manitoba Institute of Methods: Female, 8wk old Balb/c mice were repeatedly challenged with inhaled house dust mite (HDM) for Child Health, Winnipeg, 2 weeks with or without simvastatin (40mg/kg; subcutaneous), fluticasone (100μg/kg; intranasal), or fluticasone and simvastatin. Two days after final challenge and treatment we measured: lung function using a MB small animal ventilator; airway inflammation by differential cell counting of bronchoalveolar lavage (BAL); and, assessed lung histology. Results: HDM exposure induced marked cellular inflammation of the lungs that consisted of 40±8% eosinophils and 23±6% neutrophils. Simvastatin or fluticasone alone had a similar effect, reducing total inflammatory cell number by 65±6% and 65±15%, respectively. Simvastatin and fluticasone in combination had a significantly greater affect, reducing total inflammatory cell count by 71±4%; this included a 92%±2% reduction in eosinophils (p<0.01) and 85%±4% reduction in neutrophils (p<0.05). Attenuation of lung tissue inflammation was confirmed in hematoxylin-eosin stained lung sections. HDM exposure increased methacholine (25 and 50mg/ml)-induced airway resistance (Raw) by 80% and lung tissue damping (G) by 300% (p<0.001). Simvastatin and fluticasone in combination blunted the HDM-induced rise in respiratory resistance: this included 40% reduction in Raw (p<0.001) and a 68% reduction in G (p<0.001) at 25 and 50mg/ ml methacholine challenge. In contrast, simvastatin or fluticasone alone had no significant effect on the HDM-induced increase in Raw and G. Conclusion: Co-administration of simvastatin and fluticasone significantly increases the suppressive effects of either drug alone on airway inflammation or hyperreactivity. This suggests simvastatin holds promise as an adjunct medication with potential steroid-sparing effects. Coming Home: A Study of the Military Family Interactions and Relationships after Deployment Alysha Jones

Interdisciplinary This poster examines familial interactions and relationships between military veterans and their spouses Studies, University of and children after returning from foreign deployment. It has been well documented that deployment and reintegration affects familial interactions and relationships in a variety of forms such as increased stress, Manitoba, Winnipeg, distant relationships, changes in behaviour, relationship dissolution, and in some cases physical/emotional MB abuse. Although always an overly joyous occasion, post deployment is a transitional process that tends to strain the family system. . If the transitional process is not properly managed, the negative effects of this process harm family well-being, particularly for children. For instance, children tend to have lower academic scores and behavioural and emotional issues, while spouses tend to struggle reconnecting physically and emotionally with their returning spouse, as well as renegotiating their roles in the family structure. These issues are further compounded by the soldier’s struggle with mental health issues and exposure to traumatic events. Combat trauma has a tendency to amplify or escalate the maladaptive interactions with each family member, which, in turn, weakens family bonds. Five Canadian Forces families were interviewed, fifteen participants in total. Through qualitative interviews, three superordinate themes emerged: Stressors and coping skills, family dynamics, and support networks. Communication with the military member during deployment plays an integral part in the transition back into the family. Communication patterns between family members are one of the determinants in the effectiveness of role and relationship negotiation during the reunion. The effectiveness of relationship renegotiation is also aided by the family’s support system. How the Gene Induction Differences, Among a Panel of Corticosteroids, can Affect Asthma Management in the Combination Therapy? Taruna Joshi1, Malcolm Johnson2, Robert Newton1 and Mark A. Giembycz1

1Airways Inflammation Introduction: Combination therapy for asthma control involves the combination of inhaled corticosteroids Research Group, (ICSs) and beta adrenoceptor agonist and is considered to be one of the most effective asthma medications available, although, this therapy was not rationally designed. Therefore, understanding of the mechanism of Snyder Institute of effectiveness, of the combination therapy is required to design asthma medications with a better therapeu- Chronic Diseases, tic index. University of Calgary, Methods: In the present study, we used BEAS-2B airway epithelial cells transfected with a glucocorticoid (GC)-response element (GRE) reporter. After treating BEAS-2B cells with a panel of corticosteroids for 6h, in Calgary, AB and the presence and absence of an ultra long acting beta adrenoceptor agonist (Indacaterol), treated cells were 2GlaxoSmithKline harvested for the luciferase assays and RNA extractions. Harvested RNA was used to generate cDNA for the Research and real-time PCR, to study the differences in gene transcription (trans-activation) abilities of a panel of corticosteroids. Gene transactivation was selected because it is now regarded as a primary mechanism underlying Development, the anti-inflammatory effects of ICSs. Uxbridge, UK Results: Concentration-response curves were constructed to seven GR ligands to compare their relative abilities to drive GRE-dependent transcription. Five of these ligands demonstrated agonism with a rank order of potency (EC50) of fluticasone furoate (FF, 2nM) > desisobutyryl-ciclesonide (DC, 2.5nM) > dexamethasone (Dex, 11nM) = GW870086X (GW, 11nM) > mifepristone (Mif, 21nM). For our real-time PCR studies, we selected real genes induced by GR ligands and expressed by BEAS-2B cells, with anti-inflammatory potential including glucocorticoid-induced leucine zipper (GILZ), kinase inhibitor protein 2 of 57 kDa (p57kip2) and cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2). An adverse effect gene, pyruvate dehydrogenase kinase 4 (PDK4), was also studied. Indacaterol significantly augmented the expression of reporter as well as real genes. Real-time PCR data showed the expression of four different genes varied with the GR ligand used. Conclusions: Present study suggests the importance of selecting suitable GR ligand, which will transac- tivate all the necessary anti-inflammatory genes maximally but will give low induction of adverse effect genes. In other words, perhaps partial agonists of GR, exhibiting better therapeutic ratio, will be a better option in asthma control, with respect to the available full agonists. HMGB1 Modulates Extracellular Matrix Synthesis by Human Airway Smooth Muscle Cells Hessam H. Kashani1,3, Saeid Ghavami1,3, Soma Tripathi1,3, Karen A. Detillieux1,3, William T. Gerthoffer4, Helmut Unruh2 and Andrew J. Halayko1,2,3

Departments of Introduction: Airway smooth muscle (ASM) cells contribute to airway remodeling via expression and secre- 1Physiology and tion of extracellular matrix (ECM) proteins. This is, in part, driven by inflammation that includes mediators such as transforming growth factor (TGF)- 1. Damage associated molecular pattern (DAMP) proteins, such 2 β Internal Medicine, as (HMGB1) High mobility group box 1. HMGB1 is a non-histone DNA-binding protein that can regulate University of Manitoba, gene expression. HMGB1 can be released in response to stress to underpin inflammation and tissue repair. and 3Biology of In this study we tested the hypothesis that HMGB1 is expressed by and regulates ECM synthesis by human ASM cells. Breathing Group, Methods and Results: We used cultured primary and hTERT-immortalized human ASM cells. Immunoblot- Manitoba Institute of ting revealed that exogenous HMGB1 (10 ng/mL) induced significant phosphorylation of p42/p44 mitogen- Child Health, Winnipeg, activated protein kinase (MAPK) within 15 minutes - an effect that was sustained through 45 minutes. We also found that TGF-β1 (2.5 ng/mL, 0-120 hrs) promoted accumulation of secreted HMGB1 in culture MB medium. By lentiviral delivery, we induced stable expression of short hairpin RNA (shRNA) and silenced expression of endogenous HMGB1 or mammalian diaphanous 1 (mDia1), a cytoplasmic scaffold protein that is required for RAGE-mediated signaling. Lack of mDia1 markedly decreased p42/p44 MAPK phosphorylation by exogenous HMGB1. In HMGB1-silenced human ASM cells, we observed significantly reduced synthesis (total cell lysates) and secretion (culture medium) of collagen A1 and fibronectin in response to TGF-β1 (2.5 ng/mL, 0-48 hrs). Exogenous HMGB1 was not sufficient to rescue ECM synthesis in response to TGFβ1 in HMGB1-silenced cells, suggesting that intracellular, but not necessarily secreted HMGB1, regulates this response. Moreover, exogenous HMGB1 alone did not induce ECM synthesis or secretion of primary cultures ASM cells. Conclusions: In human ASM cells HMGB1 activates MAPK signaling via mDia1-dependent signaling, through an axis that likely involves RAGE. HMGB1 is released by ASM cells exposed to TGF-β1, but secreted HMGB1 does not appear to directly affect ECM synthesis, rather an intracellular role is evident. Thus HMGB1 has potential to regulate tissue repair processes involving ASM, making it a candidate for modulating airway remodeling in lung disease. Resistance to Leishmania donovani in P110d Knock-in Mice: Role of Regulatory T Cells in Liver Pathogenesis Forough Khadem, Xiaoling Gao, Zhirong Mou, Ping Jia, and Jude Uzonna

Department of Introduction: Leishmaniasis is caused by Leishmania parasites. Common forms of Leishmaniasis are: cuta- Immunology, Faculty of neous (CL), mucocutaneous and visceral leishmaniasis (VL). The annual burden of VL is increasing in endemic and non endemic regions because of global traveling, expensive and highly toxic treatments, and also the Medicine, University of emergence of drug resistance and Leishmania-HIV coinfection. Therefore, there is an urgent need to develop Manitoba, Winnipeg, new therapies/vaccines against leishmaniasis. Mammals have 3 catalytic subunits of class IA Phosphoinosit- MB ide 3 kinases (PI3Ks) with the p110δ isoform being enriched in leukocytes. Regulatory T cells (Tregs) play important roles in resistance to many pathogens. Mice with an inactivating knock-in mutation in the p110δ isoform of PI3K (p110δD910A), paradoxically are resistant to L. major (cause of CL) and mount suppressed Th1 and Th2 responses. Here, we investigate the role of PI3K in immunity to VL and the mechanism underlying it. Methods: BALB/c (WT) and p110δD910A mice were injected with 5×107 L. donovani. At different time points, parasite burden and granuloma formation were assessed in the liver and spleen by limiting dilution assay and immunohistochemistry, respectively. T and B cell responses in the liver and spleen were determined by ELISA and flow cytometry. In addition, Tregs were expandedin vivo (by injecting anti-IL-2 immune complex) and its impact on resistance was assessed by comparing parasite burden in the liver and spleens of WT and p110δD910A mice. Results: P110δD910A mice harbor significantly fewer parasites than WT mice despite having less mature granulomas and impaired T and B cell responses. Spleen Tregs in infected p110δD910A mice were lower than WT mice throughout the course of infection. Selectively expanding Tregs in vivo by anti-IL-2 immune complex dramatically abolished the enhanced resistance to VL in p110δD910A mice. Conclusion: Collectively, our results indicate that the enhanced resistance to Leishmania in the absence of p110δ signaling is independent of parasite species and is mediated in part by impaired Treg expansion in the absence of PI3K signalling. They further suggest that targeting this pathway may be useful novel approach for treatment of VL and CL. Rac1b, A Variant Of Rac1 Interacts With Calmodulin Neha Khanna, Bing Xu, Prashen Chelikani, Rajinder P. Bhullar

Department of Oral Rac1b, a splice isoform of Rac1, is up-regulated in malignant colorectal and breast cancer. It contains an Biology, University of additional 19 amino acid insertion close to the switch II domain, a region important for Rac1 interaction with regulators and effectors. This insertion leads to decreased GTPase activity and reduced affinity for GDP, Manitoba, Winnipeg, resulting in the intracellular predominance of GTP-bound Rac1b. Previously, a 14 amino acid region essential MB for calmodulin (CaM) binding has been established in Rac1. A similar region exists in Rac1b. This led to our hypothesis that Rac1b interacts with CaM. Western Blot analysis showed that endogenous Rac1b interacts with CaM-Sepharose, and that Rac1b binds to pure CaM, in a calcium dependent manner. Experiments using W7, a potent CaM antagonist, revealed that W7 does not play a role in GDP/GTP binding to Rac1b. However, the role of CaM in the activation and function of Rac1b still remains to be analyzed and is being pursued. Exploring the Role of miR-200b in Normal and Abnormal Human Lung Development due to Congenital Diaphragmatic Hernia Naghmeh Khoshgoo1,2,3, Ramin Kholdebarin1,2, Barbara Iwasiow1,2 and Richard Keijzer1,2,3

1Manitoba Institute Introduction: Congenital Diaphragmatic Hernia (CDH) is a developmental defect in the diaphragm associ- of Child Health, ated with potentially lethal pulmonary hypoplasia and hypertension due to abnormal lung development. A 2 genetic cause has not been identified, suggesting a role for epigenetics factors such as microRNAs. According Department of to our previous microarray data, miR-200b expression was significantly increased in pulmonary tissues from Surgery and fetuses with CDH. In this study we aimed to investigate the role of miR-200b in normal and abnormal lung 3Department of development via mechanisms associated with TGF-beta/SMAD signaling. Methods: We used in situ hybridization to investigate miR-200b expression and cell specific distribution in Physiology, University three human neonatal lungs tissue and age-matched controls. Also, we evaluated protein expression and of Manitoba, Winnipeg, cellular distribution using immunohistochemistry of two important targets of miR-200b: ZEB2 and TGF- MB beta2. To assess the effects of miR-200b on TGF-beta/SMAD signalling in bronchial epithelial cell (BEAS-2B) cells we transfected these cells with miR-200b inhibitors or mimics using a SMAD-luciferase reporter assay. We confirmed the results with Western blotting. Results: Semi-quatitative in situ hybridization demonstrated higher expression of miR-200b in lung tissues from patients with CDH when compared to age-matched controls. Immunohistochemistry indicated lower protein expression of ZEB2 and TGF-beta2 in CDH lungss. Luciferase assays demonstrated that miR-200b has an inhibitory effect on TGF-beta/SMAD signalling in BEAS-2B cells. Western blotting showed higher expression of p-SMAD2 and ZEB2 in miR-200b knockdown cells. Conclusions: Hypoplastic lungs in congenital diaphragmatic hernia are associated with increased expression of miR-200b that results in a decrease in TGF-beta/SMAD signaling through targeting of TGF-beta2 and ZEB2 upstream and downstream of the signaling. To our knowledge, this study demonstrates for the first time that microRNAs and specifically miR-200b is involved in abnormal lung development and congenital diaphragmatic hernia. Exploring the Role of miR-200b in Normal and Abnormal Rat Lung Development due to Congenital Diaphragmatic Hernia Naghmeh Khoshgoo1,2,3, Ramin Kholdebarin1,2, Carly Fraser1,2, Fuqin Zhu1,2, Barbara Iwasiow1,2 and Richard Keijzer1,2,3

1Manitoba Institute of Introduction: Congenital Diaphragmatic Hernia (CDH) is a developmental defect in diaphragm associ- Child Health, ated with potentially lethal pulmonary hypoplasia and hypertension due to abnormal lung development. A genetic cause has not been identified, suggesting a role for epigenetics factors such as microRNAs. Our 2 Department of preliminary results showed that miR-200b expression is significantly altered in human CDH lung tissues. In Surgery and this study we aimed to further investigate the effects of miR-200b in the nitrofen rat model for CDH and via 3Department of mechanisms associated with TGF-beta/ SMAD signaling. Methods: We studied miR-200b expression and cell specific distribution in the nitrofen-induced rat model Physiology, University of CDH and control fetal lungs by rt-qPCR and in situ hybridization (ISH). We evaluated the impact of nitrofen of Manitoba, Winnipeg, on miR-200b expression in human bronchial epithelial cells was by rt-qPCR. We then assessed the effects of MB miR-200b on TGF-beta/SMAD signalling in nitrofen treated/untreated cells transfected with miR-200b inhibitors or mimics using a SMAD-luciferase reporter assay. We confirmed the results using western blotting. Results: qPCR and ISH demonstrated lower expression of miR-200b in nitrofen-induced hypoplastic lungs and in the nitrofen treated cells. Luciferase assays demonstrated that miR-200b inhibitors or nitrofen stimulate TGF-beta/SMAD signalling and miR-200b mimics decrease the effect that nitrofen has on this signalling. Western blotting showed higher expression of p-SMAD2 and ZEB2 in miR-200b knockdown or nitrofen- treated cells. Conclusions: Pulmonary hypoplasia in the nitrofen model of CDH is associated with decreased miR-200b expression. Our results suggest that nitrofen stimulates TGF-beta/SMAD signaling via decreasing the expression of miR-200b, which result in increased higher expression of ZEB2 as one of the miR-200b targets. To our knowledge, this study demonstrates for the first time that microRNAs and specifically miR-200b is involved in abnormal lung development and nitrofen-induced lung hypoplasia. Investigation of the Kinin-Kallikrein Pathway in Prion Pathogenicity Kamilla Kosciuczyk1,2, Lise Lamoureux1, Margot Plews1, Sharon L.R. Simon1, J. David Knox1,2

1Prion Diseases It has been suggested that the conversion of the host-encoded prion protein (PrPc) to the misfolded Program, Public Health disease-associated isoform (PrPd) results in the loss of the putative superoxide dismutase enzyme activity of PrP. Another hallmark of prion diseased brains is atypical inflammation represented by presence of Agency of Canada, activated microglia capable of generating prodigious amounts of reactive oxygen species (ROS). Increased Winnipeg, MB, oxidative stress susceptibility combined with increased exposure to ROS is thought to explain the neurode- 2Department of generation observed. Previously, 2D-DIGE analysis of the urine of mice infected with the scrapie strain, Me-7, and aged matched controls demonstrated differential abundance of the proteins kininogen, kallikrein and Medical Microbiology prostaglandin H-isomerase. These proteins are all members of the kinin-kallikrein pathway suggesting its and Infectious modulation in response to prion disease. Downstream effects of the pathway produce metabolites; pros- Diseases, University of taglandins, ROS, and nitric oxide which initiate inflammation and contribute to oxidative stress, characteristics that have been demonstrated to be associated with prion diseases. It has not yet been demonstrated Manitoba, Winnipeg, whether or not the presence of these proteins in the urine, reflect disease process occurring in the primary MB site of prion pathology, the brain. Our hypothesis is that the activation of the kinin-kallikrein pathway may be in part responsible for the atypical inflammation and oxidative stress observed in the prion infected brain. To investigate, the levels of pathway-specific proteins including kininogen as well as the inducible nitric oxide synthase and the B1 receptor in the brains of ME-7 infected C57/BL6 mice, are being determined. Western blotting has demonstrated that the levels of kininogen fluctuate at the onset of clinical disease. To determine whether this is a cause or consequence of the disease the levels of 8-OHdG, a marker of oxidative stress, and glial fibrillary acidic protein (GFAP) in the brain will be measured at corresponding time points. These studies will establish whether the kinin-kallikrein pathway might be involved in the atypical inflammation and oxidative stress observed in the brain. Diminazene aceturate (Berenil) Modulates the Production of Inflammatory Cytokines by Inhibiting Phosphorylation of MAPKs and STAT Proteins Shiby Kuriakose, Helen Muleme, Chukwunonso Onyillagha, Emeka Okeke and Jude E. Uzonna

Department of Diminazene aceturate (Berenil) has been widely used as a trypanolytic agent in livestock since 1955, but Immunology, Faculty of its mechanisms of action remain poorly understood. We previously showed that Berenil treatment leads to dramatic suppression of proinflammatory cytokines (IL-6, IL-12 and TNFα) production by splenic and liver Medicine, University of macrophages and a concomitant reduction in serum levels of these cytokines in mice infected with Try- Manitoba, Winnipeg, panosoma congolense or challenged with lipopolysaccharide (LPS). In the present study, we investigated the MB molecular mechanisms through which Berenil alters inflammatory cytokine production by macrophages. We show that pre-treatment of immortalized and primary bone marrow-derived macrophages with Berenil dramatically suppressed their production of IL-6, IL-12 and TNF following LPS, CpG and Poly IC stimulation without altering the expression of toll-like receptors (TLRs) including TLR2, TLR4 and TLR9 and CD14 on macrophages. Instead, it significantly downregulated phosphorylation of mitogen-activated protein kinases (p38, ERK and JNK), and signal transducer and activator of transcription (STAT) proteins (STAT1 and STAT3) both in vitro and in vivo. In addition, Berenil also downregulated LPS-induced phosphorylation of NFkB p65 and directly suppressed IL-6 promoter activity. Interestingly, Berenil treatment upregulated the phosphorylation of STAT5 and the expression of SOCS3, a negative regulator of immune response that has been shown to inhibit phosphorylation of MAPKs and STATs. Collectively, these results show that Berenil downregulates macrophage proinflammatory cytokine production by inhibiting key signalling pathways associated with cytokine production and suggest that this drug may be used to treat conditions caused by excessive production of inflammatory cytokines. Methods for Measuring Multimorbidity Clusters using Population-Based Data John Paul Kuwornu1, Lisa M. Lix1, Shahin Shooshtari1,2

1Department of Introduction: Multimorbidity is defined as the co-occurrence of two or more chronic conditions. There is in- Community Health creasing interest in understanding the prevalence and patterns of multimorbidity because of rising chronic disease prevalence in the general population as well as in at-risk groups, such as the Aboriginal population. Sciences, University of The study objective was to describe the patterns of chronic disease that co-occur in Canada’s Aboriginal Manitoba, population and estimate the prevalence of disease clusters. 2Department of Family Methods: We used data from the 2005 Canadian Community Health Survey (CCHS) Cycle 3.1. The study cohort was restricted to adults (i.e., 18+ years) who self-identified as being of Aboriginal origin and who Social Sciences, reported having 2+ of the following common chronic conditions: asthma, arthritis/rheumatism, high blood University of Manitoba, pressure, migraine, bronchitis/COPD/emphysema, diabetes, heart disease, and mental disorders. Descriptive Winnipeg, MB statistics was used to estimate the prevalence of individual chronic health conditions. Latent class analysis (LCA) was used to identify clusters of cohort members with similar multimorbidity profiles. We used the Akaike information criterion (AIC) to identify the best fitting model. Sampling weights ensured the estimates were representative of the population. Results: The study cohort was comprised of 2,360 CCHS respondents with a mean age of 40.6 years (SD =26.5); 49.3% were males. The average number of chronic conditions was 3.8 (SD =3.3). A four-cluster LCA model provided the best fit to the data. The very low multimorbidity risk cluster (76.5% of respondents) had low prevalence (<10%) of all eight chronic conditions. The top two most prevalent conditions in the low (11.0% of respondents), medium (9.6% of respondents), and high (2.9% of respondents) multimorbidity risk clusters were mental disorders (47.6%) and migraine (47.0%), high blood pressure (73.3%) and diabetes (39.6%), and arthritis/rheumatism (84.3%) and asthma (75.9%). Females were four times more likely to be in the low and medium risk clusters than in the high multimorbidity risk cluster. Conclusions: This study revealed four multimorbidity groups within the Aboriginal population that differed in prevalence and co-occurring chronic conditions. Further research is needed to validate these clusters in other at-risk populations. Disease management programs should be designed to jointly address common clusters of chronic conditions. Duplication of Cytoband 17q25 and its Synteny Regions is a Cross-Species Characteristic of Aggressive Tumors A. Kuzyk, J. Gartner and S. Mai

Department of Cytoband 11E2 in mouse, and its human (17q25) and rat (10q32) synteny regions, is aberrant in many ag- Biochemistry and gressive types of lymphoid and non-lymphoid tumors and has been implicated as a potential oncogenic cluster important in tumorigenesis. Previously we determined that the sole aberration in aggressive fast- Medical Genetics, onset mouse plasmacytomas (PCTs) is trisomy of 11E2, which does not occur in non-aggressive slow-onset University of Manitoba, PCTs. The increased expression of genes in this region is necessary for the accelerated development of Winnipeg, MB PCTs. Furthermore, we found that the chromosome harboring 11E2 has selectively longer and protected telomeres (P = 4x10-16), illustrating the functional importance of this region for fast-onset PCT development. N-Myc levels are used to stratify human neuroblastomas, with N-myc amplified tumors characterized as more aggressive than those where it is not amplified. The most frequent chromosome change in human neuroblastoma is a gain of 17q, an indicator of poor prognosis. We hypothesize that cytoband 17q25 is the key region in chromosome arm 17q, whose duplication is necessary for aggressive neuroblastoma development. We will present data from human neuroblastoma cell lines and primary neuroblastoma patient tissue samples that illustrate an increase in the copy number of 17q25 in aggressive tumors. We have found that N-myc amplified neuroblastoma cell lines and patient tissue samples have an increased copy number of cytoband 17q25. In an aggressive human neuroblastoma cell line (IMR-32), five copies of cytoband 17q25 were observed in metaphases and 3D persevered interphase nuclei; however, a non-aggressive cell line (SH-EP/ SF), has only three copies. Aggressive neuroblastoma patient samples exhibit five and six copies of cytoband 17q25, whereas non-aggressive tumor tissue has only three copies and non-tumor lymph node tissue from the same patient has the normal two copies of 17q25. We propose that cytoband 17q25, and its synteny regions, may be a novel biomarker of aggressive tumorigenesis. ZAP-70 Increases B Cell Adhesion to Stromal Cells in Chronic Lymphocytic Leukemia: A Novel Role for CD27 Sandrine T. Lafarge1,2, Hongzhao Li1, Samantha D. Pauls3, Sen Hou1, James B. Johnston2, Spencer B. Gibson1,2,3 and Aaron J. Marshall1,3

1Department of Background: Chronic lymphocytic leukemia (CLL) can remain stable for long time periods or can rapidly Immunology, progress. Disease progression is often associated with expression of the ZAP-70 tyrosine kinase and migration of leukemic B cells (B-CLL) from the blood into lymphoid tissues such as bone marrow and lymph University of Manitoba, nodes. These tissue microenvironments enhance B-CLL survival and proliferation through a combination of 2Cancercare Manitoba, direct cell contact and soluble factors. Here we examine the impact of ZAP-70 expression on B-CLL interac- Manitoba Institute of tion with stromal cells, protective cells found in lymphoid organs. Methods: To study adhesion to stromal cells, B-CLL were cultured with mouse S17 or human HS-5 stromal Cell Biology, 3 cells with or without a pan PI3K inhibitor PI103 ± CD40L+IL4. The next day, the number of bound B-CLL to Department of stromal cells and the surface expression of adhesion molecules were determined by flow cytometry. Biochemistry and Results: We found that the ZAP-70 positive subgroup of CLL patients showed increased stromal cell adhesion and ectopic expression of ZAP-70 was able to directly enhance stromal cell binding. Inhibition of Medical Genetics, PI3K activity by PI103 or stimulation via CD40L+IL4 decreased or increased stromal cell adhesion and B-CLL University of Manitoba, survival in stromal cell co-culture, respectively. Moreover, the ZAP-70 positive patients exhibited a different Winnipeg, MB integrin expression profile, suggesting the existence of an unconventional binding system. Interestingly, we observed a higher CD27 (TNFRSF7) expression in the ZAP-70 positive subgroup and this expression is modulated by PI103 and CD40L+IL4 as well. Using a CD27 blocking antibody impaired B-CLL adhesion to stromal cells, revealing an unexpected role for CD27 in adhesion. Conclusions: Our results describe for the first time the different mechanisms that ZAP-70 positive and ZAP-70 negative B-CLL use to adhere to a supporting microenvironment. Moreover, this study highlights the novel role of CD27, a well-known marker of memory B cells, in adhesion to stroma. Histone Substrate Specificity of MYST Acetyltransferases is Selected by their Associated Factors Marie-Eve Lalonde1, Karen C. Glass2, Nikita Avakumov1, France-Hélène Joncas1, Nehmé Saksouk1, Eric Paquet1, Michael Holliday2, Song Tan3, Xiang-Jiao Yang4, Tatiana G. Kutateladze2 and Jacques Côté1

1Laval University The MYST family of acetyltransferases is required for regulation of diverse cellular pathways through epige- Cancer Research netic processes. Among this family, MOZ is known to acetylate histone H3 in chromatin, is essential for the maintenance of hematopoietic stem cells and for regulation of Hox genes expression. Another MYST, HBO1, Center, Quebec City, acetylates histone H4 and also plays an important role in embryonic development as well as in cell cycle QC, 2University of regulation through the p21 pathway. These enzymes form complexes by associating with ING tumor sup- Colorado Denver pressor proteins and with scaffold proteins that contain an EPC (Enhancer of Polycomb) homology domain; Brpf1 being the scaffold for the MOZ complex and Jade1 for the HBO1 complex. Recently, it was suggested School of Medicine, that HBO1 could also associate with Brpf1 in vivo. By performing RMN studies, peptide pull-down assays and Aurora, CO 3Center for enzymatic assays with purified complexes, we dissected Gene Regulation, The the different domains of Brpf1 for function in chromatin binding, recognition of epigenetic marks and histone acetyltransferase activity. Genome-wide location analysis of the different complexes was also perfor- Pennsylavania med using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). Unexpec- University, PA 4McGill tedly, these studies unravelled a new feature of MYST acetyltransferases, where the association of HBO1 with University, Montreal, Brpf1 redirects its activity toward histone H3 on chromatin instead of histone H4. Reciprocally, the formation QC of the MOZ-Jade1 complex changes its specificity of acetylation from H3 to H4. Furthermore, a precise dissection of the scaffold proteins allowed the discovery of a short N-term region within their EPC domain, which is responsible for selecting the histone tail that is acetylated on chromatin. These results highlight a crucial new role of the associated scaffold subunits within MYST complexes in directing the acetylation of specific histone tails, a function previously thought to reside solely within the acetyltransferase subunit. Our work shed light on the molecular mechanism by which MYST acetyltransferases bind chromatin and acetylate their substrates, thus helping to understand their role in regulating critical pathways such as gene expression, DNA synthesis and repair, cell cycle progression and differentiation. Rac1 Deficiency in the Second Heart Field Results in Congenital Heart Defects Carmen Leung1,2, Xiangru Lu1,3, Murong Liu1,3, Derek Little1, Qingping Feng1,3

1Department of Introduction: Congenital heart defects (CHD) are the most common human birth defect, affecting up to Physiology & 5% of newborns, and is the leading cause of death from congenital malformations in the first year of life. Thus, a further understanding of the mechanisms underlying CHD is crucial. The second heart field (SHF) is Pharmacology, a progenitor cell population that undergoes high rates of proliferation and migrates to contribute to the Schulich School of right ventricle (RV), ventricular septum and outflow tract of the developing heart. Rac1 is a small signaling Medicine & Dentistry, G-protein that acts as a pleiotropic effector of numerous cellular processes including proliferation, migration and survival. However, the role of Rac1 specifically in SHF progenitors during heart development remains University of Western elusive. Ontario, London, ON, Methods: We hypothesized that Rac1 is a critical regulator of SHF progenitors and a SHF-specific deletion 2Collaborative Program of Rac1 will lead to CHD. To test this hypothesis, mice with a SHF-specific deletion of Rac1 (Rac1SHF) were generated. A Rac1SHF;mT/mG global double-florescent reporter mouse was generated to lineage trace SHF in Developmental progenitors with a Rac1 deficiency. Biology, 3Lawson Results: At postnatal day one, all Rac1SHF mice exhibited extensive CHD including ventricular septal de- Health Research fects, atrial septal defects, bifid cardiac apex, overriding aorta and a thinner RV myocardium. E11.5 Rac1SHF Institute, The University had increased apoptosis in the ventricular septum and RV without any significant changes in cell proliferation. Collagen explant cultures of E12.5 Rac1SHF;mT/mG RV exhibited less migrating SHF-derived cells. of Western Ontario, E13.5 Rac1SHF RV had decreased expression of Abi1, WAVE2, Arp2 and Arp3, which are part of the WAVE and London, ON Arp2/3 complexes and act as Rac1 downstream regulators of actin polymerization. At E14.5, SHF lineages were reduced in Rac1SHF hearts. In addition, expression of the developmental cardiac transcription factors GATA4, Nkx2.5, Tbx5 and Hand2 were also significantly decreased in the RV myocardium of E13.5 Rac1SHF- mice. Conclusion: We conclude that a Rac1 deficiency in the SHF leads to complete penetrance of CHD. Our study demonstrates for the first time that Rac1 is a critical regulator of normal septum and RV formation via SHF progenitor survival, migration and developmental cardiac gene expression program during embryonic heart development. Cryopreserved Alternatively Activated Macrophages Attenuate Murine DNBS Colitis Gabriella Leung and Derek M. McKay

Gastrointestinal Introduction: We have previously established that a transfer of bone marrow-derived alternatively ac- Research Group, tivated macrophages (AAMs) can protect against DNBS-induced colitis, a model of inflammatory bowel disease (IBD). AAMs are associated with TH2 cell responses and the resolution of inflammation. As a treat- Department of ment for patients with IBD, it would be ideal for AAMs to be readily accessible for immediate therapeutic Physiology and use. Therefore we sought to determine whether cryopreserved AAMs can retain their phenotype in vitro and Pharmacology, Calvin, their anti-colitic effect in a mouse model. Methods: To generate AAMs, bone marrow cells were cultured with 20 ng/ml M-CSF for 7 days, followed by Phoebe and Joan 20 ng/ml IL-4 for 48 hrs. Cells were frozen in 10% DMSO in FBS for at least 1 week. Fresh and cryopreserved Snyder Institute for AAMs were compared on their viability (PI/Annexin V staining for flow cytometry), LPS-induced cytokine Chronic Diseases, secretion (IL-10, IL-12), and their AAM phenotype (PCR, western blotting for arginase 1, Ym1, Relm-a; arginase assay). To determine their anti-colitic effect in vivo, AAMs were injected into male BALB/c mice on day University of Calgary, -2 (10^6 in 250 μl PBS, intraperitoneal), DNBS administered on day 0 (3 mg in 100 μl 50% ethanol, intrarectal Calgary, AB 3 cm from rectum), and mice necropsied on day 3. Mice were assessed by changes in body weight, colon length, macroscopic damage, myeloperoxidase activity, and H&E colon histology. Results: Cryopreserved AAMs maintained their arginase activity in vitro, however their mRNA transcripts for canonical AAM markers were significantly decreased compared to fresh AAMs. Despite their differences in vitro, they remained protective against DNBS colitis. Collectively these data suggest that cryopreserved AAMs may be a viable option in the treatment of IBD. Resveratrol Reverses Cardiac Dysfunction in Obese Prone but not in Obese Resistant Rats Xavier Lieben Louis1,2, Sijo Joseph Thandapilly1,2, Suresh Kumar1, Liping Yu1, Carla Taylor1,2,3, Peter Zahradka1,2 and Thomas Netticadan1,2

1Canadian Centre for Objective: The objective of this study was to test the effectiveness of resveratrol in reversing cardiovascular Agri-Food Research in abnormalities in rats fed with a high fat (HF) diet. Methods: Obese prone (OP) and obese resistant (OR) were fed a HF diet for 17 weeks; Sprague Dawley Health and Medicine, (SD) rats fed laboratory chow served as control animals. OP, OR and SD rats were randomized into treated Winnipeg, MB (received resveratrol in the last 5 weeks of the study) and non-treated groups. Cardiac structure and func- 2Department of tion, blood pressure, adiposity, glucose, insulin, lipidemia, inflammatory and oxidative stress markers were assessed. Physiology, University Results: OP rats had significantly higher body weight and adiposity when compared to OR rats; treatment of Manitoba, had no effects on these parameters. HF fed OP and OR rats had prolonged isovolumic relaxation time (IVRt). 3Department of Treatment with resveratrol significantly decreased IVRt in OP, but not in OR rats. Fasting serum glucose was increased in OP and OR rats while insulin was elevated in OP rats only. Treatment reduced serum glucose, Human Nutritional while insulin levels were increased in both OP and OR rats. OP rats had increased levels of inflammatory and Sciences, University of oxidative stress markers, serum triglycerides and low density lipoprotein, which were significantly reduced Manitoba, Winnipeg, with treatment. MB Conclusions: Treatment reversed abnormalities in heart function in OP rats, but not in OR rats. Resveratrol improved several metabolic parameters such as hyperglycemia, hyperlipidemia, oxidative stress and inflammation which are also known cardiovascular risk factors in OP rats. Accordingly, the results of our study show that resveratrol reversed cardiac damage in obese settings. PgaB is Required for the De-N-acetylation and Recognition of the Biofilm Exopolysaccharide Poly-β-1,6-N-acetyl-d-glucosamine Dustin J. Little1,2, Joanna Poloczek3, Grace Li1,2, Howard Robinson4, Regis Pomes1,2, Mark Nitz3, and P. Lynne Howell1,2

1Department of Bacterial biofilms represent a significant medical problem because once established they are difficult to Biochemistry, eliminate as the bacteria are resistant to antibiotics, protected from the environment, and isolated from the innate immune system. A key exopolysaccharide required for the development and integrity of the University of Toronto, biofilm in a wide variety of pathogenic bacteria is poly-b-1,6-N-acetyl-D-glucosamine (PNAG). Four proteins, 2Molecular Structure PgaA/B/C/D, are required for the polymerization, modification, and export of PNAG in Escherichia coli. The and Function, The goal of our research is to characterize the de-N-acetylation of PNAG and how this modification targets the exopolysaccharide for export. PgaB is required for the partial de-N-acetylation of PNAG (dPNAG), and is an Hospital for Sick essential step for export. This suggests that the enzymatic activity of PgaB may be an attractive target to Children, Toronto, ON, combat biofilm formation. We have expressed, purified, and crystallized PgaB and the structure has been sol- 3Department of ved in complex with iron and nickel at 2.1 and 1.9 Å, respectively. The structure of PgaB reveals an N-terminal domain that adopts an ( / ) fold common to family 4 carbohydrate esterases and a C-terminal ( / ) Chemistry, University b a 7 b a 8 fold that is structurally similar to family 18 and 20 glycoside hydrolases. PgaB de-N-acetylase activity has of Toronto, Toronto, ON, been characterized using synthesized PNAG oligomers, and showed low catalytic efficiency with maximal 4Brookhaven National activity against a PNAG pentasaccharide, and no activity on the β-1,4-N-acetyl-D-glucosamine oligomers 2+ Laboratory, New York, of chitin. Characterization of the metal dependency showed increased de-N-acetylation activity with Co , Fe2+ and Ni2+ but not Zn2+. The C-terminal domain showed binding to PNAG oligomers using tryptophan NY fluorescence quenching assays and the structure of this domain has been solved in complex with glucosamine to 1.9 Å. These data in combination with molecular dynamic simulations of PgaB with N-acetylglucosa- mine and glucosamine suggests PNAG de-N-acetylation occurs first with subsequent binding of dPNAG to the C-terminal domain. We believe this concerted action plays a pivotal role in targeting dPNAG for export through the outer membrane porin PgaA. Antigen-Presentation of HIV-1 Gag Peptides by HLA-A*01:01 and B*07:02 Comparison of Induced Host Gene Expression Lewis Liu, Phil Lacap, Rupert Capina, Binhua Liang, Brian Fristensky, Blake Ball, Frank Plummer, and Ma Luo

National Microbiology Host Class I Human Leukocyte Antigen (HLA) presents intracellular viral antigens to direct cell mediated Laboratory, Public immunity (CMI), a critical component for controlling viral infection. Our group had previously identified two HLA class I alleles, HLA*A*01:01 and B*07:02, which are associated with reduced or increased susceptibil- Health Agency of ity to HIV-1 infection, respectively, in the Pumwani sex worker cohort established in 1985 in Nairobi, Kenya. Canada, Winnipeg, MB, HIV-1 Gag epitope analysis showed that the major difference between the protective allele A*01:01 and the Department of Medical susceptible allele B*07:02 is the binding spectrum of the Gag peptides. A*01:01 can only bind to 3 Gag peptides, while B*07:02 can present 30 Gag peptides. In this study we investigated whether the quality of epi- Microbiology, tope induced immune response is different between A*01:01 and B*07:02 epitopes by comparing peptide University of Manitoba, induced gene expression profile using PBMCs of a patient expressing both A*01:01 and B*07:02. The PBMCs Winnipeg, MB were stimulated with NSSKVSQNY peptide specific for A*01:01 or SPRTLNAWV peptide specific for B*07:02 for 8 hours. Peptide induced gene expression profile was analysed by RNA-Seq. Preliminary results showed there was no significant differences due to peptide stimulation at the current study power, however there were absolute differences in STAT3 and TLR4 signal transduction pathway. These results are being validated in a second study. Ethanol Exposure and Withdrawal Alter Global DNA Methylation Patterns and MeCP2 in Neural Stem Cells Vichithra R.B. Liyanage, Robby M. Zachariah, Carl O. Olson, and Mojgan Rastegar

Regenerative Medicine Prenatal exposure to ethanol leads to range of neurodevelopmental disorders known as Fetal Alcohol Spec- Program, Faculty of trum Disorders (FASD). Not only exposure to ethanol but also its withdrawal leads to neurological symptoms referred to as alcohol withdrawal syndrome (AWS). Recent studies show the role of epigenetic reprogram- Medicine, University of ming as a mechanism of ethanol teratogenesis and there are evidences for the role of DNA methylation Manitoba, Winnipeg, and methylation related genes such as MeCP2 in FASD. However, how ethanol affects global levels of DNA MB methylation during neural development remains elusive. Even though MeCP2, a key epigenetic regulator in the brain has been shown to be misregulated by ethanol exposure before, the mechanism by which ethanol affects MeCP2 is unknown. Therefore in this study, utilizing differentiating neural stem cells as an in vitro model system for neural development, we assessed the effects of both ethanol exposure and withdrawal on global DNA methylation levels and the expression of Mecp2/MeCP2. Our findings on the effect of ethanol on global DNA methylation levels significantly contribute to the current knowledge of the involvement of epigenetic mechanisms in ethanol teratogenesis. Our results also provide novel mechanisms for ethanol-mediated misregulation of Mecp2 during neural development. These studies provide insights on understanding the role of DNA methylation in Fetal Alcohol Spectrum Disorders and possibly design therapeutic strategies to cure the suffering children. Protective Effects of Hydrogen Sulphide in Prolonging Graft Function and Survival during Allogeneic Renal Transplantation Ian Lobb1,2, Michael Davison2, Weihua Liu1, Bertha Garcia1, Zhu Lan2 and Alp Sener1,2

1Schulich School Background: Organ procurement is inherently associated with ischemia-reperfusion injury (IRI), resulting of Medicine and from loss and subsequent restoration of blood flow and is detrimental to overall graft function and survival. Hydrogen sulphide (H S) has previously been shown to protect tissue against ischemic injury. We aimed to Dentistry, Western 2 characterize the protective role of H2S against renal IRI in a clinically applicable model of allogeneic renal University, London ON, transplantation (RTx). 2Matthew Mailing Methods: Following bilateral native nephrectomy, recipient Lewis rats underwent RTx with left kidneys obtained from Brown Norway donor rats that were flushed, at the time of procurement, with either cold (4°C) Centre for Translational standard University of Wisconsin solution (UW group) or cold UW + H2S donor molecule (150 µM NaHS) Transplant Studies, (H2S group) solution and stored for 6 hours at 4°C in the same solution. Animals were monitored for 14 days London Health or until time of sacrifice. Post-operatively, a subset of animals from each group were treated with the immu- nosuppressant Tacrolimus (Prograf®, 0.3 mg/kg; UW + TAC; H2S + TAC) daily. Serum was analyzed periodically Sciences Centre, for creatinine and blood urea nitrogen (BUN) concentrations as markers of kidney function. London, ON Results: H2S treatment significantly improved recipient survival compared to UW animals (p<0.05). Tacro- limus treated animals in both groups survived past 14 days. Whereas UW treated animals exhibited high serum creatinine and BUN levels that remained elevated until time of death, H2S treated animals initially exhibited reduced serum creatinine and BUN levels compared to UW that gradually increased until death later in the time course. Both Tacrolimus groups initially exhibited slightly elevated serum creatinine and BUN levels which returned to normal by day 7. Conclusions: These findings are the first to report that2 H S protects kidney allograft survival and function. H2S treatment appears to delay graft rejection and may present a novel clinical solution to improve the efficacy of kidney transplantation. CFTR Mediates Bacteria-triggered Mucus Secretion by Airway Submucosal Glands in Swine Xiaojie Luan1, L. Dean Chapman2, and Juan P. Ianowski1

1Departments of Background: Cystic fibrosis (CF), the most common, fatal genetic disease affecting young Canadians, caused Physiology and by mutated CFTR anion channel. CF lung disease is currently the source of most morbidity and mortality in CF patients. CF airway disease reflects the failure of the innate defense mechanisms of the lung against in- 2 Anatomy and Cell haled pathogenic organisms such as the bacterium Pseudomonas aeruginosa. Normal airways are protected Biology, College of from inhaled `insults’ by a complex immune defense system that involves mucus and antimicrobial factors Medicine, University of secretion. The mucus traps bacteria that are killed and/or inactivated by antimicrobial factors and finally removed from the airway by mucociliary clearance. Most mucus and antimicrobial factors secretion in the Saskatchewan, upper airways is produced by submucosal glands. In CF airways, submucosal gland secretion seems to be Saskatoon, SK abnormal. Thus, it has been suggested that abnormal mucus secretion in CF airway may be the pathogenic event that triggers a sequence of infection and inflammation that eventually may lead to respiratory failure. However, the response of airway submucosal glands to inhalation of bacteria has never been directly tested. Objective: To test whether inhaled bacteria triggers CFTR-mediated mucus secretion and to investigate the role of the proinflammatory cytokines interleukin (IL)-1b, IL-6 and tumor necrosis factor (TNF)- a in this process. Methods: We use synchrotron-based imaging to measure the mucus layer in isolated swine trachea. We also use electrophysiological and secretion essay techniques to investigate the effect of proinflammatory cytokines on mucus secretion in isolated preparations. Results: Our results show that P. aeruginosa stimulate mucus secretion by submucosal gland in isolated tracheas. This secretion only occurred in the vicinity of the bacteria but not the whole airway. Also, our results show that CFTR is required for bacteria-stimulated mucus secretion. Interestingly, flagellin, an abundant structural protein from the flagellum on bacteria, is required to produce bacteria-triggered mucus secretion. Literature shows Flagellin-TLR5 interaction in the airways triggers cytokines secretion, and we have evidence showing that the proinflammatory cytokines IL-1β, IL-6, and TNF-a may be involved in the bacteria-triggered mucus secretion, which directly stimulated the Na+ and Cl- transports at serous acini of the submucosal glands. Statistical Analysis of Tracheal Breath Sounds during Wakefulness for Screening Obstructive Sleep Apnea Cameron A. MacGregor, Davood Karimi, Ali Azarbarzin, Zahra Moussavi

Department of Obstructive sleep apnea (OSA) is a prevalent disorder. The accepted method of diagnosis in widespread Electrical and clinical practice, polysomnography (PSG), is costly and very time consuming; therefore, quick screening methods, especially when there is a need for quick diagnosis, is of great interest. Diagnostic methods which Computer Engineering, exploit subtle differences in breath sounds recorded during wakefulness, such as our group’s Awake-OSA University of Manitoba, technology, have shown their capability to diagnose OSA at the research stage. Simplifying the breath Winnipeg, MB sound recording procedure employed in the Awake-OSA diagnostic method would increase its efficiency when used in a clinical setting. In this study, we adopted breath sound data collected during wakefulness in two positions (sitting upright and supine) and two breathing maneuvers (nose and mouth breathing) from our previous study [10], and ran statistical analysis on a wide variety of sound features to select the most significant features correlated with OSA. The goal was to investigate which combinations of patient position and breathing maneuver contribute the least to the significant features between groups of people with differing OSA severity, thus permitting simplification of the recording protocol. The results showed that all signals recorded by a combination of the two breathing maneuvers and two positions resulted in some features significantly correlated with OSA severity, making it impossible to confidently recommend that a combination be omitted from the recording protocol. Nevertheless, the results show that the majority of significant features originated from recordings made in the supine position. Therefore, as a step toward simplification of the Awake-OSA diagnostic algorithm, we may use breath sound signals recorded only in the supine position and further investigate the accuracy of the algorithm in distinguishing amongst groups with differing OSA severity. Analyses of Natural Killer Cell Migrations in a Real-time and Dynamic Microfluidic System Sajid Mahmood1, Saravanan Nandagopal2, Francis Lin2 and Sam K.P. Kung1

1Department of Introduction: Bidirectional interactions between mature natural killer (NK) cells and dendritic cells (DC) Immunology and require cell-cell contact and cytokine signals that occur in lymphoid and non-lymphoid tissues. Such interactions are important in coordinating innate and adaptive immune responses that shape anti-tumor and 2 Department of anti-microbial responses in vivo. The role of this critical association in regulating NK trafficking, however, is Physics, University of still not well characterized in vitro. Manitoba, Winnipeg, Methodology: The conventional methods used in cell migration assays (such as the transwell method) lack the ability to provide stable gradient environment for single cell level analysis. Emerging microfluidic device MB based approach has been developed to provide better control over stable gradient generation and to mimics complex microenvironment for the quantitative analyses of chemotaxis, chemokinesis and, potentially, chemorepulsion at a single cell level. In the present study, we established a novel real-time, live cell imaging microfluidic platform in the studies of IL-2 activated NK cells migrationin vitro. We generated immature and lipopolysaccharide (LPS)-matured bone marrow derived DC (BMDC). Culture supernatant of these DC cultures was collected and assayed for its ability to regulate NK migration in the microfluidic system. Results: We found that soluble factors released by immature and LPS-matured BMDC contained chemotactic signals which induced respectively, a modest and high level of chemotactic movements of IL-2 activated NK cells in vitro. We confirmed these findings in a standard transwell migration assay and identified that CXCR3 is a key receptor on NK cells that mediated the migration. More interestingly, using this microfluidic system, we observed also chemorepulsion of NK cell migration for the first time. Conclusion: This microfluidic system should prove useful in elucidating factors that regulate NK trafficking behaviour and in studying NK migration in microenvironments that mimics inflamed peripheral sites. Possible Neuroprotective Function of miRNAs Upregulated during Pre-Clinical Prion Disease Anna Majer1,2, Yulian Niu1 and Stephanie A. Booth1,2

1Department of Background: Currently, the molecular process governing synaptic dysfunction and neuronal loss during Medical Microbiology, prion-induced neurodegeneration remains largely unknown. Investigating molecular changes that occur early in disease, when the infectious prion protein is beginning to accumulate, may hold promise in iden- University of Manitoba, tifying these disease-related pathways. To this end, we performed extensive high-throughput transcriptomic Winnipeg, MB, 2Public and miRNomic temporal screens on a neuronal-rich brain region (CA1 hippocampus). From this screen, we Health Agency of identified the presence of a neuroprotective response that occurred during early, pre-clinical disease and diminished as disease progressed. This protective mechanism was evident not only at the transciptomic Canada, Winnipeg, MB level but also at the microRNA (miRNA) level. In fact, out of the 7 miRNAs that were upregulated during early prion disease, 3 miRNAs have known neuroprotective functions. We hypothesize that the remaining 4 dere- gulated miRNAs also contribute to the neuroprotective process. Methods: Real-time PCR and in situ hybridization for the 4 candidate miRNAs (miR-16, miR-26a, miR-140 and miR-146a) was used to further validate the expression levels of these miRNAs in prion infected CA1 hippocampal regions. MiRNA target prediction programs compared with mRNA data were employed to discern neuronal specific genes regulated by these miRNAs. Currently, we are performing Luciferase reporter assays to validate the top candidate targets. To identify potential effects of these miRNAs on neuronal morphology, over-expression and knock-down experiments for each miRNA will be performed in primary mouse hippocampal cultures. Results: We confirmed the upregulation of 4 miRNAs in early prion-induced neurodegeneration using real-time PCR and in situ hybridization techniques. Based on bioinformatic predictions, targets of these 4 candidate miRNAs were strongly associated with neuronal function. Currently, we have successfully optimized our primary mouse neuronal cultures and we are in the process of testing the effects of these miRNAs in affecting neuronal morphology (i.e. dendrite complexity and spine morphology). Conclusions: We have shown that numerous miRNAs were involved in a neuroprotective program long before clinical symptoms were apparent in an animal model of prion disease. We believe that miR-16, miR- 26a, miR-140 and miR-146a also exhibit neuroprotective properties and their contribution to this protection remain the focus of further study. A Causal Role of Dorsolateral Prefrontal Cortex Neurons during a Feature-Based Visual Working Memory Task Nour Malek, Roberto Gulli, and Julio C. Martinez-Trujillo

Department of Visual working memory (WM) is the temporary storage of visual information that is no longer available to Physiology, McGill one’s sight but that is relevant to a task at hand and that is necessary for reasoning and planning. Deficits in WM have led to the cardinal cognitive and motivational syndromes plaguing brain disorders such as University, Montreal, Alzheimer’s, stroke, and schizophrenia. Visual WM can be segregated into two forms: spatial—involved with QC remembering locations of stimuli, or feature— associated with recalling specific features of stimuli (e.g., motion direction, orientation). While several neuroscience laboratories have engaged in the search for the neural correlates and mechanisms of WM in several brain areas, most have come to agree that the dorsolateral prefrontal cortex (dlPFC) is central to WM processes. Moreover, spatial WM networks in dlPFC have been extensively investigated and have yielded computational models of WM; the same cannot be said for feature WM, without which render the generated models incomplete. Of primary interest is whether WM coding, spatial- or feature-based, in dlPFC dictates behavioral response. This causal nature has already been observed in healthy human subjects whose WM performance was reversibly disrupted via transcra- nial magnetic stimulations (TMS) of the dlPFC. However, similar implications have yet to be replicated on the fundamental neuronal level. Utilizing a delayed match-to-sample task, which consists of matching the feature—motion direction—of a sample random dot pattern (RDP) moving in a unilateral direction to 1 of 2 test RDPs, and optogenetics, we hope to make such insights. Optogenetics is a reversible means of modulating neuronal activity using light-sensitive ion channels. Two types of lentivirus vectors would be injected into the dlPFC of a Macaca mulatta to make neurons sensitive to light: (1) the excitatory channelrhodopsin-2 (hCHR2) and (2) the inhibitory archaerhodopsin from Halorubrum (ArchT). Once expression in dlPFC neurons is achieved, we will: (1) record their normal, single unit activity, without stimulation, via single electrodes and determine each neuron’s selectivity; and (2) couple the single electrodes with optic fibers and a laser—which would shine a blue or yellow light on the neuron to excite or inhibit it, respectively—to record dlPFC neuron activity during stimulation. Upon analyzing reaction times and the animal’s performance, we anticipate that inhibiting dlPFC neurons would deteriorate memory, while exciting them would enhance memory. This experiment’s benefits are two-fold. First, the neural activity observed during the delay periods (i.e., the memory periods) of this task with and without stimulation could provide clarity to progressing computational models for feature-based WM. Finally, determining such a causal link or the lack of one for dlPFC on WM would further the understanding of the pathophysiology of WM deficits in brain disorders and could promote the development of different intervention strategies. S100A4 signaling in Thyroid Cancer Manoj R. Medapati1, Mathias Dahlmann4, Ulrike Stein4, Saeid Ghavami2, Alok Pathak3, and Sabine Hombach-Klonisch1

1Department of Introduction: Thyroid cancer (TC) is one of the most frequently occurring cancers of endocrine glands in Human Anatomy, Canada and the United States. Most differentiated TC are curable, but some TCs develop a more aggressive phenotype with resulting metastasis and treatment failures. Our group has shown that relaxin-like peptides 2 Department of promote TC cell tumor growth and migration, in part mediated via S100A4 which is known to promote me- Physiology, tastasis and to confer poor prognosis in patients with TC. We showed that S100A4 is secreted by TC cells. 3Department of Hypothesis: Extracellular S100A4 signals via the receptor for advanced glycation end products (RAGE) in TC cells and promotes TC migration. Surgery, University of Material & Methods: Immunofluorescence, immunohistochemistry, Western blot analysis and RT-PCR were Manitoba, Winnipeg, performed to study the expression of RAGE transcripts and protein in TC primary cells, TC cell lines and TC MB, 4Max-Delbrück- tissues. RAGE pull-down experiments were performed to investigate S100A4 binding to RAGE in TC cells. TC cells were stimulated with recombinant human S100A4 to identify S100A4-induced signaling pathways by Center, Berlin, Germany Western blot. RAGE silencing using RAGE siRNA and lentiviral shRNA knock-down of diaphanous-1, the intracellular signaling partner of RAGE, was performed to investigate the requirement for RAGE in S100A4-signaling. TC migration upon S100A4 was determined using two-chamber cell migration assays. Results: We showed expression of S100A4 and RAGE transcripts in human TC cell lines and human thyroid primary cells. In thyroid carcinoma tissues, RAGE protein was localized exclusively in TC cells, adjacent non-cancerous thyroid cells were devoid of RAGE protein. In vitro pull down assays demonstrated that S100A4 binds to RAGE in TC cells. We identified JNK/SAPK, MAPK and NFkB signaling pathways to be predominantly activated in TC cell lines by extracellular S100A4. Knock down of Dia-1 suppressed S100A4-induced ERK signaling indicating RAGE to be the mediating receptor for extracellular S100A4. Silencing of Dia-1 also prevented the S100A4-mediated increased migration of TC cells. Does the Increase in BMI Associated with Initiation of Insulin Therapy among Youth Newly Diagnosed with Type 1 Diabetes Predict Overweight or Obesity at age 18? Taru Manyanga and Randy Fransoo

Department of Setting: Overweight and obesity in youth have increased over time. Childhood overweight and obesity Community Health are associated with both short-term and long-term negative health outcomes. It is well documented that initiation of insulin therapy in type 1 diabetic patients can lead to an immediate increase in Body Mass Index Sciences, University of (BMI). No published studies have examined if this BMI increase persists to adulthood. Manitoba, Winnipeg, Objectives: 1. To examine if an increase in BMI at 6 or 12 months after initiation of insulin therapy in youth MB with type 1 diabetes is associated with overweight or obese status at age 18. 2. To examine the relationship with other clinical markers related to cardio-metabolic health (HbA1c, dyslipidemia, hypertension, creatinine and albumin). Methods: Data from the Diabetes Education Resource for Children and Adolescents (DER-CA) database housed at the Children’s hospital will be used for this study. This unique database combines extensive clinical information on each patient with virtually universal coverage. The study population will comprise all children and youth diagnosed with type 1diabetes in Manitoba between 1986 and 2012. Regression models will be used to assess the association between change in BMI at 6 and 12 months after diagnosis, and overweight or obesity status, in addition to the other clinical markers listed in objective 2 above. The models will control for sex, age at diagnosis, BMI and clinical markers at diagnosis, and insulin regimen. Additional stratified analyses will examine sub-groups within the sample, to determine whether the effects are different for children with different characteristics (e.g. initial BMI, age at diagnosis, or sex). Results: Pending Potential Implications: If this study finds a significant association between post-diagnosis weight gain and deleterious outcomes, then clinical practice may need to be modified to monitor and control weight gain associated with initiation of insulin therapy. The Development of Novel HIV Vaccine Vectors: A Cynomolgus Macaque Cytomegalovirus-Based Approach Angie K. Marsh1, David O. Willer2,3, Aruna P.N. Ambagala2,3, Misko Dzamba4, Jacqueline K. Chan2,3, Richard Pilon5, Jocelyn Fournier6, Michael Brudno4, Paul Sandstrom5 and Kelly S. MacDonald1,2,3

1Department of Within the field of HIV vaccine design, it has been recognized that there is a real need to diversify the pool Immunology, University of vectors undergoing testing and to utilize vectors that can induce durable immunity. Herpesviruses, of Toronto, such as cytomegalovirus (CMV), show much promise as viral vectors as they establish lifelong latency with 2Department of periodic reactivation enabling persistent immune boosting of vaccine antigens. We propose that using Medicine, University of herpesviruses as lifelong reactivating HIV viral vectors will generate a robust and persistent effector memo- Toronto, 3Department ry T-cell response at the mucosa, which is the site of HIV entry and may impair HIV replication at its earliest stage. In an effort to evaluate CMV-based HIV/SIV vectors in a non- human primate (NHP) model, we isolated of Microbiology, Mount and characterized the complete genomic sequence and organization of a novel cynomolgus macaque CMV Sinai Hospital, Toronto, (CyCMV; Ottawa strain) using next-generation sequencing. The CyCMV genome is 218,041 bp with 84% ON, 4Department of protein-coding density and 262 putative ORFs. We compared and contrasted the structural and functional Computer Science, genes of CyCMV to human CMV (HCMV) with respect to pathogenesis, immune evasion, and species-spe- University of Toronto, cificity. Furthermore, to examine evolutionary differences between CyCMV strains of different geographic 5 Toronto, ON, National origins, we have recently completed the genomic sequencing of a CyCMV strain isolated from a cynomolgus HIV & Retrovirology macaque from the Mauritius Island for comparison to CyCMV (Ottawa strain) isolated from a macaque of Laboratories, Public Filipino origin. In order to maximize the ability to manipulate the virus for vaccine purposes, we are cloning Health Agency of CyCMV as a Bacterial Artificial Chromosome (BAC). To initiate cloning of CyCMV-BAC, we generated a recom- Canada, Ottawa, ON binant CyCMV-GFP virus using homologous recombination. Following plaque purification of CyCMV-GFP, 6Scientific Services the BAC was introduced via Cre-lox recombination. The cloning of CyCMV-BAC has enabled the virus to be Division, Health transformed into a bacterial system for future manipulations and the insertion of vaccine antigens. This Products & Food newly characterized CMV provides a novel model in which to study HIV/SIV CMV-based vaccines in hopes of Branch, Health Canada, informing forthcoming human clinical trials, and in addition CyCMV-BAC will be accessible for CMV biolo- Ottawa, ON gists allowing for virus manipulation studies (insertions, deletions, mutations), functional characterization of CMV genes and furthermore HCMV vaccine development. Building the Base: Challenges, Successes and Next Steps Towards Implementing Evidence-Based Mental Health Screening for Prisoners. Michael Martin

Department of Introduction: High rates of mental illness in jails and prisons have been consistently documented in Epidemiology, cross-sectional prevalence studies. However, a number of studies have raised concerns about the ability to detect mental illness in prison settings. As a result mental health screening at the beginning of incarceration University of Ottawa, now underpins almost all best practice guidelines and prison mental health strategies. Ottawa, ON Methods: This poster presents the author’s involvement in a series of projects to develop evidence-based mental health screening for prisoners. First, an Iterative Classification Tree scoring model was developed to increase the efficiency of mental health screening in Canadian prisons. Second, a systematic review was conducted to compare alternative screening options to the process offered within Correctional Service of Canada (CSC). Results: The introduction of mental health screening in Canada and in other countries has led to improved detection of mental illness, but with significant over-referral. Initial results show that the new scoring model implemented by esc has improved the efficiency of screening. Conclusion: Screening is a critical component to a correctional mental health strategy. While replication studies are required, there appear to be some improvements in screening in recent years. However, high rates of over-referral may draw resources away from providing treatment to those with the highest need. An increased understanding of the impacts of screening on mental health and behaviour throughout incarceration and upon release to the community is needed to determine the appropriate balance of false positives and negatives at the screening stage. Bfp Pilin-Like Proteins Form Part of the Structure of the Bundle Forming Pili in Enteropathogenic Esherichia coli E2348/69 Claudia F. Martínez de la Pena1, George Mulvey1, Leon De Masi2, Michael Donnenberg2, and Glen Armstrong1

1Department of Enteropathogenic Escherichia coli (EPEC) is one of 6 different diarrheogenic pathotypes of E. coli. EPEC use Microbiology, type IV pili (T4P) called Bundle-forming pili (BFP) for initial adherence to the host cell, a step that is crucial for colonization. In addition to bundlin, the main BFP structural protein, several pilin-like proteins, BfpI, BfpJ, Immunology, and and BfpK, are also encoded in the BFP operon. Since BFP are produced on the bacterial surface and are im- Infectious Diseases, portant for host colonization, and because pilin-like proteins have been found to be minor subunits of other 2Division of Infectious T4P systems, it is important to know the role of these pilin-like proteins in BFP function and biogenesis. To this end, we cloned BfpI, BfpJ, and BfpK into the pQE31 vector (Qiagen) which produces 6His-tag N-termi- Diseases, University of nal protein fusions. The BfpI, BfpJ and BfpK proteins were then used to produce specific rabbit polyclonal Calgary, Calgary, AB antisera. Rabbits were injected with 100ug of purified protein mixed with complete Freund’s adjuvant and boosted two more times with incomplete Freund’s adjuvant. The polyclonal antibodies were used with goat anti-Rabbit IgG conjugated to 15nm gold nanoparticles to perform Immunogold Electron microscopy. Briefly, EPEC strains were grown on blood Agar plates and EPEC colonies were re-suspended and placed on a formvar/carbon nickel grids, blocked and incubated with 1:100 dilutions of the polyclonal anti-BfpI, BfpJ, or BfpK or pre-immune sera. After washing the samples were incubated with secondary antibody, then washed and stained with 1% phosphotungstic acid. The grids were viewed using a Hitachi H-7650 Transmission Electron Microscope. We found that in the EPEC strain E2348/69 the proteins BfpI and BfpJ are present in the structure of the BFP filament. The use of double mutants with the retraction ATPase (BfpF) and pilin-like proteins have helped to elucidate the role of Bfp proteins when single mutants did not express pili. Using the double bfpF bfpI mutant we found that this strain is not able to perform Localized Adherence to Hep-2 cells at early time points (45 min), suggesting that this protein will have some role in the pilus structure or directly in the binding with host receptors. The role of BfpJ and BfpK in the pilus structure and function is being further investigated. Frontline Healthcare Workers’ Attitudes Towards a Future HIV Vaccine for Vulnerable Communities in Karnataka, South India Leigh McClarty1, Satyanarayana2, Robert Lorway1, John Wylie1,3, Marissa Becker1,3,4

1Department of Background: Approximately 2.4-million people in India live with HIV. The southern state of Karnataka has Community Health the nation’s fifth-highest HIV prevalence (0.63%). HIV disproportionately burdens Karnataka’s female sex workers (FSWs) and men who have sex with men (MSM). The best long-term strategy for managing the glo- Sciences, University of bal HIV epidemic likely involves an effective vaccine; however, the availability of a vaccine will not guarantee Manitoba, 2Karnataka its accessibility or acceptability. The objective of this study is to gain an understanding of the barriers and Health Promotion facilitators to delivery and uptake of an HIV vaccine among these communities. Methods: We conducted an exploratory cross-sectional survey among healthcare workers (HCWs) in Kar- Trust, Bangalore, India, nataka working with MSM/FSWs in relation to HIV prevention, care and support. Interviewers administered 3Department of face-to-face questionnaires. We used multistage, random sampling with stratification by district, target po- Medical Microbiology, pulation, type of clinic/organization, and type of HCW. Descriptive statistical analysis was carried out using SPSS 20.0. University of Manitoba, Results: 379 HCWs participated in the study. Mean age of participants was 35.3 ± 10.5y; females comprised 4 Department of 64.6% of the sample. More HCW participants were members of the MSM/FSW communities than not (56% Internal Medicine, vs. 44%). A significant majority of participants felt they were very knowledgeable about HIV (74.7%). Only University of Manitoba, 38.3% knew that HIV vaccines were being tested in clinical trials. 292 participants (77%) reported that nothing would prevent them from recommending an HIV vaccine to MSM/FSWs. Yet, a significant majority Winnipeg, MB admitted to being uncomfortable recommending the vaccine to boys and girls < 13 y (80.0% and 72.8%, respectively). Findings varied by HCWs’ socio-demographics and job type. Implications: This study contributes to the limited vaccine acceptability literature focusing on marginalized groups and the knowledge gap pertaining to frontline HCWs’ attitudes towards a future vaccine. Despite reporting an overall willingness to recommend an HIV vaccine to MSM/FSW community members, these findings imply that HCWs in Karnataka might have underlying hesitations to deliver an HIV vaccine to certain target groups within the community. Such discrepancies warrant further exploration and understanding in order to ensure optimal future HIV vaccine roll out to most at risk groups. Molecular Characterization of Rotavirus Isolates from Select Canadian Pediatric Hospitals Andrew McDermid1,2, Nicole Le Saux3, Elsie Grudeski1, Julie Bettinger4, Kathy Manguiat1, Scott A. Halperin5, Lily MacDonald1, Pierre Déry6, Joanne Embree2,7, Wendy Vaudry8, and Timothy F. Booth1,2

1National Microbiology Introduction: One of the major causes of childhood gastroenteritis is rotavirus. Rotavirus causes roughly Laboratory, Winnipeg, MB, 453,000 deaths annually, primarily in the developing world. In Canada, rotavirus disease causes a substantial 2Department of Medical amount of burden and expense on the healthcare system. Rotavirus contains a double stranded segmented Microbiology, University of RNA genome frequently used for genotype surveillance. The G (VP7) and P (VP4) genome segments used Manitoba, Winnipeg, MB, genotyping encode the outer capsid proteins which are the basis for two rotavirus vaccines. This study 3Division of Infectious Diseases, CHEO Research focuses on genetic surveillance of rotavirus strains circulating in Canada from 2007-2010 and predictions of Institute, Children’s how vaccines will provide protection against circulating strains. Hospital of Eastern Methods: Stool samples from eight Canadian pediatric hospitals were collected and sent to the National Ontario, Ottawa, ON, Microbiology Laboratory for genetic surveillance. From 2007 to 2010, 348 samples were collected and tested 4Vaccine Evaluation using heminested multiplex PCR. A subset of samples was used for full genome analysis, which involved Center, BC Children’s comparison of circulating rotavirus amino acid sequences within antigenic motifs found in vaccines. Hospital, University of Results: Surveillance showed 174 G1P[8], 45 G3P[8], 22 G2P[4], 13 G9P[8], 3 G4P[8] and 2 G9P[4] rotavirus British Columbia, Vancouver, BC, 5Canadian circulating in Canada. Genotype frequency fluctuated year to year and site to site. Upon analysis of antigenic Center for Vaccinology, motifs there were several mismatched amino acid residues when comparing circulating strains with those IWK Health Center and composing currently licensed vaccines. Dalhousie University, Conclusion: Genetic surveillance from this study showed that circulating strains have a very similar geno- Halifax, NS, 6Centre Mere- typic profile to that found in available vaccines. Analysis of antigenic amino acid motifs of the VP4 and VP7 Enfant de Quebec, CHUL, proteins suggests that circulating rotavirus strains have evolved since vaccine strains circulated. Considering and Laval University, both the surveillance data and the amino acid motif analysis, the currently licensed vaccines will likely be Quebec City, QC, sufficient to substantially reduce rotavirus morbidity in Canada when vaccines are universally funded and 7Winnipeg Children’s Hospital, Winnipeg, MB, uptake is increased. However, given the changing profile of rotavirus genotypes in Canada, the recently 8Stollery Children’s discovered G9P[4] strains and the amino acid motifs of the antigenic regions of the surface proteins there is Hospital and University of potential for vaccines to escape vaccine acquired immunity in the future. As more provinces fund rotavirus Alberta, Edmonton, AB vaccines, it will be important to monitor vaccine effectiveness with continued surveillance. Conditional Sox9 Ablation Post-Spinal Cord Injury Reduces Chondroitin Sulfate Proteoglycan Expression and Improves Motor Function William M. McKillop, Elisa York, Andreas Schedl, and Arthur Brown

Robarts Research The lack of axonal regeneration following human central nervous system injury is attributed in part to glial Institute, The University scarring. This glial scar is produced primarily by reactive astrocytes and consists of many anti-regenerative molecules chief of which being the chondroitin sulphate proteoglycans (CSPGs). Since we have previously of Western Ontario, identified Sox9 as a transcription factor that up-regulates the expression of CSPGs post spinal cord injury London, ON (SCI), and that ablation of Sox9 prior to SCI leads to improved locomoter recovery post SCI, we predicted that a delayed conditional Sox9 ablation 1 week post SCI (a more clinically relevant time point) would be sufficient to reduce CSPG expression and improve locomotor recovery post SCI. We investigated the effect of delayed Sox9 knock-down on glial scar composition and motor function recovery after 70 kdyne spinal cord contusion injury delivered to thoracic level 9. Neurological recovery in Sox9flox/flox;Cre mice and Sox9flox/flox littermates was assessed for 14 weeks post spinal cord injury (SCI). One week after injury mice were administered tamoxifen to ablate Sox9. We report that in comparison to mice expressing normal levels of Sox9, delayed Sox9 conditional knockout mice display reduced levels of CSPG core proteins (brevican, neurocan and aggrecan), and GFAP (a marker of astrocyte activation) in the injured spinal cord compared to controls 6 weeks post SCI. These changes in gene expression were accompanied by improved hind limb function and locomotor recovery as evaluated by the Basso Mouse Scale (BMS) and rodent activity boxes at 14 weeks post-injury. Histological assessments confirmed reduced expression of GFAP, CSPGs, and peri-neuronal networks in Sox9 conditional knock-out mice, and demonstrated increased serotonin immunoreactivity cau- dal to the lesion epicenter 14 weeks post SCI. The increased avenues for plasticity and improved functional recovery following 1 week delayed Sox9 knockout highlights the clinical potential of an anti-Sox9 treatment for SCI. Multiplex Molecular Imaging in-vivo Using an Ultra-Small Clinical Endoscope Patrick Z. McVeigh1, Israel Veilleux2, Brian C. Wilson1,2

1Department of Introduction: As a whole, medicine is undergoing a fundamental shift from systemic imaging and therapy Medical Biophysics, to a more target-based approach, enabled by the explosion of disease-specific biomarkers being discovered each year. While standard detection techniques such as fluorescence and chemiluminescence are suitable University of Toronto, for biomarker imaging in-vitro, they cannot be easily translated to in-vivo imaging due to poor probe sta- Toronto, ON, 2Ontario bility and high endogenous background levels found in human tissue. Nanoparticle contrast agents which Cancer Institute/ generate signal based on surface-enhanced Raman scattering (SERS) offer the ability to image multiple (5+) biomarkers simultaneously at concentrations which are orders of magnitude lower than what is possible University Health with fluorescence. The ability to perform SERS imaging endoscopically would provide a unique platform for Network, Toronto, ON in-situ cancer screening or procedural guidance. Methods: A library of spectrally distinct SERS-active nanoparticle probes has been created using targeting monoclonal antibodies which are capable of detecting an expression ‘fingerprint’ of non-small cell lung cancer (NSCLC). These multifunctional nanoparticles are validated on the target tumour cell lines using flow cytometry and ex-vivo tumour staining. A world-first ultra-small scanning fiber endoscope (1.2mm outer diameter) was modified to permit direct SERS imaging in the near infrared (NIR) to minimize the background from tissue. The sensitivity and multiplex imaging capability of the endoscopic SERS system was tested on stained NSCLC samples in both the in-vitro and in-vivo settings. Results: Conjugation of targeting antibodies to SERS nanoparticle imaging agents did not significantly affect the specificity of the targeting agent. The resultant particles were found to be significantly more stable in-vivo than comparable fluorescent probes and showed no sign of photobleaching over 3 hours of conti- nuous imaging. The direct SERS imaging system has a linear response from (0.1-100pM) and was capable of 4x simultaneous quantitative imaging without compensation for spectral overlap. Widefield quantitative SERS imaging in a mouse model of NSCLC in-vivo had 4x multiplex biomarker probe quantification which was in excellent agreement with known quantities (R2>0.98). Conclusions: Molecularly-targeted contrast agents based on SERS can provide the ability for multiplexed, quantitative imaging in-vivo which is orders of magnitude more sensitive than what is possible with fluorescence. Endoscopic SERS imaging is possible at picomolar biomarker concentration levels in-vivo and represents a significant opportunity in personalized cancer imaging and therapy. Regulation of Cardiolipin Remodeling in Human Lymphoblasts Edgard M. Mejia1,4, William A. Taylor1,4, Marilyne Vandel1,4, and Grant M. Hatch1,2,3,4

1Department of Background: Cardiolipin (CL) is a major phospholipid found exclusively in mitochondria and plays an Pharmacology and important role in the biogenesis and dynamics of mitochondrial membranes and a key role in mitochondrial energy production. Functional CL is highly enriched with linoleic acid. Barth Syndrome (BTHS) is a rare Therapeutics, X-linked genetic disorder caused by mutations in the TAZ gene. The TAZ gene product, tafazzin, remodels de 2Biochemistry and novo synthesized cardiolipin (CL) with linoleic acid. TAZ mutations result in cardiomyopathy and neutrope- Medical Genetics, nia. Monolysocardiolipin acyltransferase-1 (MLCL AT-1) is an enzyme that is also capable of remodeling CL with fatty acids including linoleic acid. 3Center for Research Objective: To examine ifMLCL AT-1 complements tafazzin in the remodeling ofCL in human lymphoblasts. and Treatment of Methods: Epstein-Barr virus transformed lymphoblasts from an age-matched healthy individual (identifier Atherosclerosis and 3798) and a BTHS patient (identifier 618) were transfected with TAZ and/or MLCL AT-1 RNAi or an MLCL AT-1 carrying plasmid. TAZ and MLCL AT-1 gene expression were analyzed in these cells as well as CL mass, MLCL 4Manitoba Institute of AT-1 enzyme activity and incorporation of radiolabeled precursors into CL. In addition, mitochondrial com- Child Health, University plexes were examined using Blue Native PAGE, in-gel activity assays and western blot analysis. of Manitoba, Winnipeg, Results: MLCL AT-1 gene expression was increased when TAZ expression was reduced in both normal and 14 MB BTHS lymphoblasts. Expression of MLCL AT-1 increased CL levels, [1- C)linoleic acid incorporation into CL and MLCL AT-1 enzyme activity in both normal and in BTHS cells in which TAZ expression was reduced. Double knockdown of both TAZ and MLCL AT-1 did not lead to a further decrease in [l- 14C) linoleic acid incorporation into CL, MLCL AT-1 enzyme activity or CL mass than TAZ knockdown alone. In addition, MLCL AT-1 expression elevated mitochondrial complex I formation and activity in both normal and BTHS lymphoblasts implicating enhanced mitochondrial function in these cells. Conclusion: MLCL AT-1 expression may serve as a potential therapeutic approach to treat BTHS. FOXP3+ T Regulatory Lymphocytes Counteract Angiotensin II-Induced Blood Pressure Rise and Vascular Injury Muhammad Oneeb Rehman Mian1, Tlili Barhoumi1, Marie Briet1, Adriana Cristina Ene1, Asia Rehman1, Pierre Paradis1 and Ernesto L. Schiffrin1,2

1Vascular and Introduction: Innate and adaptive immunity participate in part via the action of macrophages and T Hypertension Research lymphocytes in the pathogenesis of angiotensin (Ang) II-induced hypertension and vascular injury. Ang II-induced hypertension and endothelial dysfunction are blunted in T and B lymphocyte-deficient (Rag1-/-) Unit, Lady Davis mice, and restored with reconstitution of T but not B lymphocytes. Recently, we demonstrated that adop- Institute for Medical tive transfer of T regulatory lymphocytes (Treg), a subset of T lymphocytes that exert immune modulating Research and actions, protected against Ang II and aldosterone-induced blood pressure (BP) rise and/or vascular injury. However, it is unknown whether Treg play a role in Ang II-induced vascular injury. We hypothesized that 2 Department of adoptive transfer of Scurfy T lymphocytes that are deficient in a critical transcription factor necessary for Medicine, Sir Mortimer Treg maturation and function, FOXP3 (also known as Scurfin), compared to wild- type cells will exacerbate B. Davis-Jewish General Ang II-induced vascular damage in Rag1-/- mice. Design and Methods: Eleven-week old male Rag1-/-mice were injected i.v. with phosphate buffered saline Hospital, McGill (PBS) containing 2% FBS (control), 107 wild-type or Scurfy pan T lymphocytes, and 2 weeks later underwent University, Montréal, sham surgery or were infused with Ang II (490 ng/kg/min, s.c.) using mini-osmotic pump for 14 days (n=3-8). QC Systolic BP (SBP) and diastolic BP (DBP) were measured by telemetry. Endothelial function and vessel structure were assessed in second order mesenteric arteries by pressurized myography. Cryosections of aorta were used to assay reactive oxygen species (ROS) production by dihydroethidium staining and monocyte/ macrophage infiltration and fibronectin expression by immunofluorescence. Collagen content was -exa mined by Sirius red staining in paraffin sections of aortas. Results: Ang II induced a 40 mmHg SBP rise in Rag1-/- mice for all treatment groups, but DBP rise was ~10 mmHg greater for wild-type and Scurfy T cell-injected mice than for control mice (P<0.01). Adoptive transfer of WT T cells restored Ang II induced-impairment of mesenteric artery endothelium-dependent relaxation to acetylcholine (P<0.05), which was exaggerated in Scurfy T cell-injected mice (P<0.01). Ang II induced a greater increase in ROS production in aortic perivascular fat of Scurfy T cell-injected mice compared to WT T cell-injected mice (P<0.05). Ang II increased stiffness of mesenteric arteries of control mice and Scurfy T cell- injected mice but to a lower extent in WT T cell-injected mice (P<0.01). Ang II treatment induced hypertrophic remodeling in control mice and Scurfy T cell-injected mice, but not in wild-type T cell-injected mice (P<0.05). Ang II increased fibronectin content to a greater extent in the aorta of control and Scurfy T cell-injected mice compared to WT T cell-injected mice (P<0.01). There was greater expression of collagen I and III in the aorta of control and Scurfy T cell-injected mice compared to the aorta of WT T cell-injected mice (P<0.01), but expression was not affected by Ang II treatment. Preliminary results indicated that Ang II increased monocyte/macrophage infiltration in PBS- and Scurfy T cell-injected mice (P<0.05), which also tended to increase in WT T cell-injected mice. Conclusion: These findings extend our previous findings that Foxp3+ T regulatory lymphocytes have a protective role against Ang II-induced vascular remodeling. Progesterone Resistance in Endometriosis: Case of IL1 Receptor Inhibitors N. Michaud and A. Akoum

Laboratory of Endometriosis is a gynecological disease characterized by the development of ectopic endometrial tissue Endocrinology of area with an abnormal synthesis of estrogen (E2) and resistance to progesterone (P4). Our research revealed a deficiency of natural IL-1 inhibitory receptor type 2 (IL1R2) and its accessory protein soluble (sIL1R3) in the Reproduction, endometrium, peritoneal fluid and serum of patients with endometriosis phase cycle dependent. These re- Research Center CHU sults indicate a possible link between ovarian hormones (E2 and P4) and IL1 system in endometriosis. Thus, Quebec-Saint Francois our hypothesis is that abnormalities in hormonal responses are involved in the IL1 system dysfunction and, consequently, in the pathogenesis of endometriosis. Hospital, Faculty of We studied the influence of ovarian hormones on the expression of IL-1 receptors in endometrial cells of Medicine, Laval women with endometriosis or normal. To do this, an analysis of the regulation of gene and protein expres- University, Montreal, sion of these receptors by quantitative real-time PCR (qRT-PCR), ELISA and Western blot was performed. In endometrial stromal cells of normal women, E2 and P4 increase the expression of IL1R2, both at the level QC of mRNA and protein (p <0.05). In women with endometriosis, by cons, E2 and P4 don’t regulate mRNA or protein of the receptor. In contrast, gene and protein expression of IL1R3 is reduced in patients than controls but increased in endometriosis. These results are consistent with original and promising progesterone resistance process described in endometriosis. In light of these results, a study of receptor type A, B and C (PR-A, PR-B, PR-C) of progesterone was performed on the same patients. Regulation of PR-B, which mediates the effects of progesterone by ovarian hormones and dominance of PR-A, which regulates the functions of PR-B was observed in the control patients (p <0.05). Whereas in patients with endometriosis no hormonal regulation and no dominance of PR-A is identifiable. In all likelihood, PR-C is not expressed in stromal cells of women with and without endometriosis. These results suggest a regulatory hormone-inflammatory mechanism ever described in the literature. Understanding such interactions is of considerable interest since they could play an important role in the development of this disease and the manifestation of his major symptoms. The Anorexigenic Neuropeptide Nesfatin-1 Influences the Excitability of Neurons in the Nucleus of the Solitary Tract Andrea Mimee and Alastair V. Ferguson

Department of Introduction: Obesity and its associated comorbidities continue to impose a severe burden on health care Biomedical and systems worldwide, and thus efforts to understand the complex neurocircuitry and neuropeptides governing the control of food intake are under intense investigation. One such peptide is nesfatin-1 (nes-1), an 82 Molecular Sciences, aa product of the nucleobindin-2 (NUCB2) gene with potent anorexigenic, cardiovascular and glucoregula- Physiology Graduate tory effects. Immunohistochemical studies have revealed the expression of nes-1 throughout the brain and, Program, Queen’s in particular, in nucleus of the solitary tract (NTS), a critical medullary autonomic gateway. In order to elucidate the mechanisms by which nes-1 exerts its central effects, the present study was undertaken to explore University, Kingston, the cellular correlates of nes-1 actions in the medial NTS (mNTS). ON Methods: Whole cell current and voltage clamp recordings were performed on single rat mNTS neurons in slice preparation. Following completion of the recording, cytoplasm was aspirated into the recording pi- pette, and mRNA was reverse transcribed and subjected to a multiplex polymerase chain reaction (RT-PCR). Results: Bath applied 10 nM nes-1 directly influences the excitability of the majority of mNTS neurons (n =93) by eliciting either depolarizing (mean 7.8±0.8 mV, 42%) or hyperpolarizing (mean 8.2±1.0 mV, 21%) responses. These effects occur in all electrophysiologically defined cell types in the mNTS, and are site specific and concentration dependent. Using single cell RT-PCR, we demonstrate a depolarizing action of nes-1 on cells involved in the control of ingestive behavior, namely neuropeptide Y and NUCB2 expressing mNTS neurons. Furthermore, 10 nM nes-1 increases the frequency and amplitude of IPSCs in mNTS neurons, suggesting pre- and post-synaptic modulation of inhibitory neurotransmission in these cells by nes-1. Conclusions: Our electrophysiological results are the first to demonstrate direct cellular actions of nes-1 on NTS neurons, while our RT-PCR findings suggest the potential for a self-stimulatory nes-1 feedback loop in these cells. Furthermore, our voltage clamp findings reveal a modulatory role of nes-1 on inhibitory synaptic transmission in the NTS. Thus, this study provides critical insight into the circuitry and physiology involved in the effects of nes-1, and highlights the NTS as a potentially key structure mediating these central actions. In vivo Regulation of T Cell Respiration and Function by Stomatin-like Protein-2 Panagiotis Mitsopoulos1, Julie St-Pierre2, Stanley D. Dunn3, Joaquín Madrenas1

1Department of Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein identified in a proteome analysis of glyco- Microbiology and lipid-enriched membrane microdomains of mammalian cells, and modulates T cell activation (J. Immunol. 2008; 181:1927, PLoS ONE 2012; 7:e37144). To study SLP-2 function we generated T cell-specific SLP-2 knoc- Immunology, McGill kout (SLP-2 T-K/O) mice since conventional SLP-2 knockout mice were embryonic lethal at the pre-implanta- University, tion stage. These mice showed reduced CD4+ T cell responses and decreased respiratory complex I and II+III 2Department of activities. Our mechanistic studies indicate that SLP-2 recruits prohibitins to cardiolipin-enriched domains in the mitochondrial inner membrane to form specialized cardiolipin-enriched membrane microdomains, Biochemistry, required for optimal cellular respiration (Mol. Cell. Biol. 2011; 31:3845). We hypothesized SLP-2 functions in Goodman Cancer this manner to regulate the bioenergetics of T cells, and that systemic deletion of the SLP-2 gene will cause Research Centre, McGill defective mitochondrial function that may result in organ-selective disease. Seahorse XF analysis revealed that although SLP-2 T-K/O cells showed unaltered total cellular and mitochondrial oxygen consumption University, Montreal, rates (OCRs), they had significantly greater non-mitochondrial OCR, mitochondrial OCR uncoupled from ATP 3 QC, Department of synthesis, and a greater reliance on glycolysis (71% of mice) (J. Immunol. 2012; 189:4349). Surprisingly, SLP-2 Biochemistry, T-K/O cells exhibited greater spare respiratory capacity, a phenomenon not due to differential sensitivities of University of Western cells to uncoupling agents. To study the function of SLP-2 systemically, we generated viable tamoxifen-inducible systemic SLP-2 knockout mice. Administration of tamoxifen (3mg/40g; 10 days; i.p.) resulted in a motor Ontario, London, ON phenotype characterized by poorer SHIRPA behavioural scores, significant weight loss and mortality (68% at day 28, p<0.05). This phenotype correlated with SLP-2 gene deletion and decreased protein levels in most tissues. Histological analysis revealed a multifocal myofibrillar degeneration of the diaphragm with a mononuclear inflammatory infiltrate. In addition, apoptosis throughout the exocrine pancreas and pancreatic atrophy due to loss of normal acinar tissue were observed. This new immunometabolic phenotype suggests SLP-2 may regulate optimal assembly of functional respiratory supercomplexes. Indeed, our preliminary evidence using two-dimensional blue native gel electrophoresis on primary T cells, brain and skeletal muscle tissue suggests SLP-2 is mainly associated with high molecular weight mitochondrial lipid-protein complexes that may correlate to respiratory supercomplexes. Bioenergetic and biochemical analyses of mitochondria are underway to establish the regulatory role of SLP-2 in the metabolism of T cells and other tissues including skeletal muscle. In conclusion, in vivo SLP-2 deletion induces a mitochondrial disease- like phenotype, suggesting this protein is required for optimal metabolic function, including that of adaptive immune responses. SLP-2-deficient mice may be useful to assess the function of SLP-2in vivo and to study mitochondrial diseases. Critical Role of Semaphorin 3E in Allergic Airway Disease Hesam Movassagh1, Lianyu Shan1, Abeer Saati1, Jonathan Duke-Cohan2, Andrew Halayko3, Jamila Chakir4, and Abdelilah Soussi-Gounni1

1Department of Introduction: Asthma is a chronic inflammatory disease of the airways contributing to bronchial hyperresponsiveness and airflow limitation. Pathophysiological features of asthma include eosinophil-rich inflam- Immunology, matory cell infiltrates, mucus hypersecretion, and airway wall remodeling. In allergic asthma, inhalation of University of Manitoba, allergens induces Th2 immune deviation and recruitment of inflammatory cells into the lungs which lead to 2Laboratory of tissue damage. The current therapies fail to overcome pathological events of asthma efficiently. Therefore, Immunobiology, development of an effective strategy to control asthma is an urgent health issue. There are some regulatory mechanisms which are abrogated in asthmatic conditions and identification of them may provide novel tar- Department of Medical gets to control the disease. Semaphorins, originally discovered as neuronal guidance cues, are multifaceted Oncology, Dana-Farber proteins expressed in many tissues and regulate cellular processes such as cell growth and migration which Cancer Institute, are impaired in asthma. Semaphorin 3E (Sema3E) negatively regulates function of some inflammatory and structural cells and it may play an important role in asthma. Boston, MA, Methods: We investigated expression of Sema3E in asthmatic and healthy individuals using immunohisto- 3Department of chemistry (IHC), ELISA and real-time PCR. Then, we characterized the effect of Sema3E on airway hyperres- Physiology, University ponsiveness (AHR), inflammation and remodeling using a mouse model of the disease by performing lung mechanics studies, flow cytometry, ELISA, etc. Sema3E We further addressed the role of Sema3E by allergen of Manitoba, Winnipeg, exposure in Sema3E KO vs WT mice. MB, 4Centre de Results: The study demonstrated that Sema3E expression is suppressed in asthmatic patients and experi- recherce de l’Institute mental animal model. Surprisingly, exogenous Sema3E treatment intranasally inhibited allergen-induced universitaire de airway inflammation, remodeling and AHR. Sema3E knockout (KO) mice developed an exaggerated form of allergic airway disease compared to wild type (WT) control group. cardiologie et de Conclusion: Our data suggests a regulatory role for a member of semaphorin family, Sema3E, on airway pneumologie du homeostasis which is impaired in asthmatic conditions. According to the results of this study and also our Quebec, Université previous findings about repulsive effect of Sema3E on airway smooth muscle cells, this molecule might be considered as a novel option to minimize airway inflammation, AHR, and remodeling in chronic airway Laval, Quebec City, diseases such as asthma. QC miR-301a Over-Expression in Bone-Marrow Derived Mesenchymal Stem Cells: Differentiation versus Proliferation Alison L. Muller1, Jessica Klassen1, Shannon Neumann1, Mina Guirgis1, Melanie Ngo1, Yun Li1, and Darren H. Freed1,2

1Department of Introduction: Bone marrow-derived mesenchymal stem cells (MSCs) have been implicated in a variety of Physiology, cardiovascular diseases, from cardiac fibrosis to in-stent restenosis and transplant vasculopathy, predominantly through the acquisition of a myofibroblast phenotype. We have previously shown that MSCs display 2 Department of a myofibroblast phenotypein vitro in addition to increased miRNA-301a expression. In this context, the Surgery, University of mechanism of MSC-myofibroblast differentiation is unclear; however, miRNA expression has been shown Manitoba, Winnipeg, to influence differentiation of other cell types. We therefore hypothesize that miRNA expression changes coincide with and influence the MSC to myofibroblast transformation. MB Methods: MSCs were isolated from the sternum of patients undergoing open heart surgery and were cultured in standard DMEM/F12 with 20% FBS. Cells were transfected using 50nM concentrations of control and pre-miR-301a. MSC structure and function was assessed with collagen gel contraction and MTT assays, immunofluorescent staining, qRT-PCR and western blotting. Results: miR-301a expression was decreased in MSCs possessing myofibroblast characteristics, compared to less differentiated cells. Compared to scramble controls, overexpression of miR-301a resulted in reduced collagen gel contraction and increased proliferation. miR-301a over-expression also decreased non-muscle myosin IIa, smooth muscle myosin and the myofibroblast marker ED-A fibronectin. Conclusions: Our results suggest a mechanistic role for miR-301a in regulating the proliferative versus differentiated phenotype of bone marrow-derived mesenchymal stem cells. Further experiments are required to determine a potential therapeutic role for miR-301a. HMG-CoA Reductase Inhibitors Impair Survival of Human Mesenchymal Stem Cells via NF-kappaB Signaling Alison L. Muller1, Yun Li1, Melanie Ngo1, Kiran Sran1, Rakesh C. Arora2, Lorrie A. Kirshenbaum1 and Darren H. Freed1,2

1Department of Introduction: Circulating progenitor cells of bone marrow origin known has mesenchymal stem cells Physiology (MSCs) have been implicated in transplant cardiac allograft vasculopathy (CAV) and cardiac fibrosis. HMG- CoA reductase inhibitors (statins) have been shown to impair the progression of CAV and improve patient 2 Department of survival. We examined the in vitro effects of two lipophilic statins (atorvastatin and simvastatin) and one Surgery, University of hydrophilic statin (pravastatin) on the viability of MSCs and expression of NF-κB and mevalonate signaling. Manitoba, Winnipeg, Methods: MSCs were isolated from the sternum of patients undergoing open heart surgery and cultured in standard DMEM/F12 with 20% FBS. MSCs were treated with atorvastatin, simvastatin, and pravastatin at MB 0.1, 1.0 or 10 μM ± mevalonate. Cell viability was assessed using an MTT assay. NF-κB p65 expression was assessed by western blot. Activation of the NF- κB pathway was achieved through adenoviral overexpression of Ikk-β. Results: MSC treatment with 1 and 10 μM simvastatin or atorvastatin resulted in progressively reduced cell viability to about 50%, corresponding with a NF-kappaB p65 decline to 70 and 50% respectively. Viability was rescued by co-incubation with mevalonate or by pretreatment with Ikk-β. Pravastatin did not affect MSC viability or NF-κB expression. Conclusions: Mevalonate depletion through HMG-CoA reductase inhibition impairs the viability of primary human MSC through down-regulating NF-κB. This represents another pleiotropic effect of statins and may explain a lipid-independent beneficial effect of this therapy. The differential effect of lipophilic versus hydrophilic compounds requires further investigation. Microfluidic Assisted Investigation of T Lymphocyte Migration in Lymph Node Relevant Chemokine Gradients Saravanan Nandagopal and Francis Lin

Departments of Background: Chemokines CCL19, CCL21 and their receptor CCR7 are known key mediators for T Physics and Astronomy, lymphocyte recruitment to lymph nodes (LNs) and navigation within LNs . However, a critical mechanism is missing to facilitate T cell exit from the T cell zone (TCZ). Previous reports demonstrated that the journey of University of Manitoba, the T cells from the TCZ to the medulla is not controlled by S1P-S1P1 interaction nevertheless arresting the Winnipeg, MB S1P-S1P1 interaction blocks the entry of the T cells into the medullary sinuses indicating there must be a possible mechanism that down-regulates CCR7 signals and brings the T cells from the TCZ to the vicinity of the egress port where S1P-S1P1 signalling guides the T cells for their ultimate exit. Hypothesis: Based on our previous experimental and modeling studies, we hypothesis that LNs sub- regional CCL19 and CCL21 fields mediate T cell exit from the TCZ through CCR7 signaling. Methods: In the present study, we analyzed CCL19 and CCL21 distribution profiles in different LNs sub- regions of mouse LNs sections by immuno-fluorescent staining and confocal microscopy, and our results conceptually support the hypothesized LNs sub-regional CCL19 and CCL21 fields. Furthermore, we established an in-vitro model to quantitatively examine the migration of activated human blood T cells in simulated LNs sub-regional CCL19 and CCL21 fields followed by cell surface CCR7 expression analysis (by immunofluorescent and Confocal microscopy) using a previously described microfluidic system. Results: Our results suggest that CCL19 and CCL21 mediate T cells migration in LNs sub-regions. In particular, the results suggest the sequential actions by specific co-existing CCL19 and CCL21 fields in the TCZ periphery and in the region further beyond for mediating T cell exit from TCZ, which is correlated with altered surface CCR7 expression. The Net Healthcare Costs Associated with Clostridium difficile Natasha Nanwa

Pharmaceutical Background: The cost-effectiveness of interventions (e.g., vaccination) related to Clostridium difficile (a Sciences, University of serious gastrointestinal infection) may not be well established, as Canadian cost estimates associated with C. difficile suffer limitations: 1) they are not centered on population based data, instead derived from Toronto, Toronto, ON assumptions or expert opinion; and 2) net costs are not calculated, possibly inflating the true cost of the disease. Objective: To estimate the net healthcare costs associated with C. difficile from the Ontario Ministry of Health and Long-Term Care (MOHLTC) perspective. Methods: This study consists of two parts. First, the study employs a retrospective matched cohort design. Health administrative data held at the Institute for Clinical Evaluative Sciences (ICES) will be used to establish a cohort of C. difficile infected subjects. Infected subjects will be included if they have an ER visit or hospital admission coded as the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, Canada code A04.7 (enterocolitis due to C. difficile). These infected subjects will then be matched to uninfected subjects using a combination of hard and propensity score matching. The second part employs a retrospective incidence-based cost of illness analysis also using the health administrative data held at ICES. The following resources will be captured, as the MOHLTC covers their costs: physician visits; outpatient laboratory and diagnostic tests; outpatient medication use; ER visits; same day surgery; procedures; hospitalizations; homecare; rehabilitation; and long-term care. Costs of the uninfected subjects will be subtracted from those of the infected subjects and then averaged across all subjects to estimate the mean net healthcare costs per infected subject. Preliminary Results: From January 1, 2003 to December 31, 2010, we identified~ 37,622 infected subjects (mean = 4,703 per year) with a mean age (± standard deviation, range) of 70 (± 19, 0-107) years and 56% female. Eleven percent (n = 4,044) had an ER visit as their initial C. difficile event (87% coded as the main diagnosis), while 89% percent (n = 33,578) had a hospital admission as their initial C. difficile event (25% coded as the most responsible diagnosis). HMGA2-Mediated Chemoresistance in Human Cancer Cells Involves ATR/CHK1 Signaling Suchitra Natarajan1, Sabine Hombach-Klonisch1, Peter Droege2, and Thomas Klonisch1

Departments of Introduction: High Mobility Group AT-hook protein-2 (HMGA2) is a non-histone chromatin-binding protein, 1Human Anatomy and expressed in embryonic stem cells, absent in normal adult cells but re-expressed in cancer cells. HMGA2 enhances chemoresistance to tumor cells by playing a role in Base Excision Repair (BER) (1). Cell Science, Faculty of Hypothesis: HMGA2 protects cancer cells from DNA damage-induced cell death. Medicine, University of Methods: Human cancer cell models expressing HMGA2 [undifferentiated thyroid cancer, A549 lung Manitoba, Winnipeg, cancer, HT1080 Fibrosarcoma and Rhabdomyosarcoma] were treated with DNA-damaging Methyl Methane Sulfonate (MMS) for 30min and then let to recover in fresh media for about 48 h. Total CHK1, phospho(p) MB, 2School of CHK1S296, total ATR and phospho(p)ATRS428 were detected by Western blot and cell cycle profiling was done Biological Sciences, by cell cycle FACS. Apoptosis was measured by luminescent caspase3/7 activation assay and protein-protein Nanyang Technological interactions were determined by co-immunoprecipitation. Results: We investigated the association of HMGA2 with DDR signaling factors, CHK1 and ATR. Upon University, Singapore MMS insult, HMGA2 clones displayed significantly lessγ -H2AX foci and this coincided with shorter comet tails. We observed significantly increased (p<0.0001) phosphorylation of CHK1 and ATR upon MMS insult and sustained presence of pCHK1 and pATR during the recovery period was dependent on the presence of HMGA2. CHK1 knockdown in MMS-treated HMGA2+ cancer cells resulted in a significant increase in apoptosis, which was enhanced further by knockdown of HMGA2. Coinciding with the prolonged presence of pCHK1 was a prolonged G2/M arrest up to 24 h of recovery. In addition, HMGA2 was found to be a novel interacting partner with ATR and CHK1. Conclusion: Our results suggest that, in addition to its role in BER, multifunctional HMGA2 (i) causes sustained activation of CHK1 signaling, (ii) prolongs G2/M arrest (iii) inhibits apoptosis and (iv) interacts with members of the ATR signaling cascade. Hence development of small inhibitors attenuating HMGA2 function may be a novel therapeutic strategy to target HMGA2+ cancers. Estimating Completeness in Administrative Databases for Ascertainment of Parkinson’s Disease: Application of Capture-Recapture Methods Y. Nie and L.M. Lix

Department of Introduction: Parkinson’s disease (PD) is a chronic and progressive movement disorder that affects quality of Community Health life and health care utilization. Population-based administrative health databases (AHDs) are widely used for PD surveillance. Capture-recapture methods (CR) have been proposed to describe the completeness of AHDs Sciences, Faculty of for estimating disease population size. However, the assumptions of conventional CR methods, homogeneity Medicine, University of of capture probabilities and independence of data sources, are unlikely to be satisfied in AHDs. Manitoba, Winnipeg, Objective: To apply CR methods based on the multinomial logistic regression model, which account for potential heterogeneity in capture probabilities, to evaluate the completeness of AHDs for estimating PD MB prevalence. Methods: We applied a multinomial logistic regression CR model to PD diagnoses in physician billing claims and hospital discharge abstracts from Saskatchewan for the 2008/09 fiscal year. A base regression model, which included covariates of sex and age group, was compared to a full model, which also contained income quintile and residence location (i.e., rural/urban), to estimate the number of PD cases missed by AHDs. Crude prevalence estimates were calculated and a bootstrap technique was used to estimate 95% confidence intervals (CIs). Results: A total of 1817 PD cases were identified from diagnoses in AHDs, with 1537 cases from physician billing claims only, 70 cases from hospital discharge abstracts only, and 210 cases from both sources. Estimated total PD cases from the base model were 1890 (95% CI: 1874, 1908), giving an estimated prevalence of 182.9 per 100,000 population (95% CI: 181.3, 184.6). The estimated cases from the full CR model were similar and the latter model did not result in a significant improvement in model fit as judged by the Akaike Information Criterion (AIC; AIC=1871.1 for full model and 1872.0 for base model). Conclusion: Regression-based CR models resulted in higher PD prevalence estimates than a conventional case ascertainment method for AHDs. The use of a simple multinomial logistic regression CR model to address heterogeneity improved the estimates over single counting methods, but additional covariates did not result in higher estimates. Techniques such as latent class analysis should also be explored to address violations of CR model assumptions. High Mobility Group Box 1 (HMGB1) Promotes Phases of Wound Healing in Bronchial Epithelial Cells O.O. Ojo1,3, S. Ghavami1,3, A. Jha1,3, M.H. Ryu1,3, Helmut Unruh2,4, Andrew J. Halayko1,2,3

1Department of Background: HMGB1 is a damage associated molecular pattern (DAMP) family member that binds toll-like Physiology, University receptors (TLR2 and TLR4) and the receptor for advanced glycated end products (RAGE). HMGB1 expression is associated with chronic airway inflammation, however, its effect directly on airway structural cells is of Manitoba, understudied. Epithelial responses are fundamental drivers of asthma, including abnormal repair-restitution 2Department of linked to extracellular matrix (ECM) synthesis, and persistence of cell proliferation and migration. In this Internal Medicine, study we tested the hypothesis that HMGB1 induces a pro-migratory phenotype that is coupled with ECM synthesis in bronchial epithelial cells. University of Manitoba Methods: Human bronchial epithelial cells (BEAS-2B) were cultured in LHC-9 media then subjected to 3Biology of Breath scratch wounding after which wound closure was measured (48hrs) by phase contrast microscopy. We Group, Manitoba tested the impact of HMGB1 (0.3-3ng/ml) on wound closure in the presence or absence of molecular and pharmacological inhibitors of RAGE (neutralising antibody 20μg/ml), HMGB1 (glycyrrhizin 100μM) or TLR4 Institute of Child (anti-TLR4 20μg/ml or TAK242 1μM). We used immunoblotting to assay ECM (fibronectin, laminin-5) andα 3 4 Health, Section of integrin abundance, as well as phosphorylation of ERK1/2, SMAD2 and JNK. Thoracic Surgery, Results: HMGB1 increased wound closure by ~30% compared to untreated controls. This effect was similar University of Manitoba, to that induced by TGFβ1 (10ng/ml). Enhanced wound closure by HMGB1 was attenuated by co-treatment with neutralizing antibodies for TLR4 or RAGE. HMGB1 also induced rapid accumulation of fibronectin, Winnipeg, MB laminin-5 and α3 integrin from 3-24hrs. Upregulation of fibronectin could be diminished by blunting TLR4 signalling, blocking RAGE, or by inhibiting extracellular HMGB1. Similarly, laminin-5 expression induced by HMGB-1 was diminished by the TLR4 signalling inhibitor, TAK242, and by the ERK inhibitor, U0126 (10μM). HMGB1 induction of α3 integrin receptor for fibronectin and laminin-5 was also blocked by TLR4 and RAGE inhibitors. Immunoblotting revealed that HMGB1 induced a rapid/sustained phosphorylation of ERK1/2 and JNK, but a delayed and short phosphorylation of SMAD2, indicating they are signalling effectors of HMGB1 receptor mediated effects. Conclusion: Our findings suggest a role for HMGB1 in human airway epithelial cell function in repair and restitution via pathways mediated by TLR4 and RAGE that underpin increased ECM and ECM receptor expression and modulate cell-cell adhesion. The p110d Isoform of PI3K Modulates TLR4 Signaling and Promotes Survival in Experimental Sepsis Emeka Okeke, Ifeoma Okwor, Ping Jia, and Jude Uzonna

Department of Sepsis, a systemic immune response to severe bacterial infection, is a leading cause of death in critical Immunology, care medicine caused by dysregulated immune response to bacteria endotoxin LPS which is recognised by TLR4. P110d isoform of PI3K is expressed mostly on leukocytes and has been shown to be important in University of Manitoba, leukocyte recruitment to sites of inflammation and infection. We report that pharmacological or genetic Winnipeg, MB inhibition of p110d signalling led to increased expression of TLR4 upon LPS stimulation an effect which was not observed for p110aand p110b. Differential cell counting showed higher numbers of inflammatory cells in the peritoneum of p110d knock-in [p110d(D910A)] mice compared to wild type mice. Macrophages from p110d(D910A) mice induced higher proinflammatory cytokines compared to WT mice in response to LPS. In addition, absence of p110d activity led to mortality in an otherwise non-lethal dose of LPS accompanied by exaggerated production of proinflammatory cytokines. We authenticate our findings by showing that p110d(D910A) mice are unable to recover from a non-lethal dose of Escherichia coli infection. Thus modulation of p110d signalling is a potential therapeutic target in acute inflammation and sepsis. Role of LIGHT, a Member of the Tumor Necrosis Factor Superfamily in Secondary Immune Response to Leishmania major Ifeoma Okwor1, Gui-Lian Xu2, and Jude E. Uzonna1,2

1Department of Different members of the tumor necrosis factor superfamily (TNFSF) are important for processes such as cell Medical Microbiology, survival, apoptosis and regulation of the immune system. We previously showed that LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T University of Manitoba, lymphocytes) or TNFSF 14 and its receptors are important for initiation of CD4+ T helper (Th1) cell response 2Department of and development of primary immunity to Leishmania major infection in mice. Here, we further characterized Immunology, the contributions of this molecule in dendritic cell (DC) maturation, maintenance of L. major-induced IL-12 production and secondary Immunity to Leishmania major. We show that blockade of LIGHT significantly University of Manitoba, impaired DC maturation, expression of costimulatory molecules, number of cells recruited into the draining Winnipeg, MB lymph nodes as well as early IFN-γ production by CD4+ T cells following L. major infection. Furthermore, we show that LIGHT is required at both priming and maintenance stages of anti-Leishmania immunity. Interestingly, blockade of LIGHT had no effect on delayed-type hypersensitivity (DTH) response and control of parasite burden following secondary L. major challenge in wild type (WT) mice. In contrast, blockade of LIGHT signaling in CD40 KO led to lower DTH and higher parasite burden. Collectively, our results show that LIGHT is critical for priming and maintenance of Th1 response during primary immune response. However, it is dispensable during secondary anti-Leishmania immunity in the presence of functional CD40 but becomes important in the absence of CD40 signaling. HIV Exposed Seronegative Women have Higher Cellular Activation and Proinflammatory Cytokine Responses to Toll-Like Receptor 7 and 8 Stimulation compared to Susceptible Women W.R. Omange1, R.-C. Su1, A.F.A. Meyers1,2,4, M. Kimani2, J. Kimani1,2, A. Maingi2, J. Nyakio2, C. Mesa3, F.A. Plummer1,2,4 and T.B. Ball1,2,3,4

1Department of The innate immune system through TLR7 and 8 has been shown to recognize the single strand RNA from Medical Microbiology- the HIV virus. The HIV virus is deadly, butnot all exposures to this virus results in infection. This evident in a small group of commercial sex workers who are thought to be repeatedly exposed to HIV virus due to University of Manitoba, their high risk sexual behaviour, without being infected. We hypothesize that a higher recognition and 2Department of responsiveness to ssRNA from HIV is a distinguishing feature of HESN phenotype. This higher recognition Medical Microbiology- and responsiveness is dependent on a modulation of the interaction between TLR signaling pathways and cytokine signaling pathways. To test this hypothesis, we stimulated freshly isolated PBMCs overnight with University of Nairobi, Imiquimod (TLR7) and ssRNA (TLR7/8), followed by quantification of surface activation markers on T cells Kenya, 3Department of and NK cells by flow cytometry and quantification of soluble cytokines by multiplex bead arrays. Finally, we Immunology- compared TLR and cytokine signaling pathways in ssRNA and Imiquimod stimulated PBMCs from HESN and NN women. University of Manitoba, The results of this study revealed differences in activation of T and NK cell subsets and in activation of Winnipeg, MB, T and NK cells of HESN and NN women after ssRNA (TLR7/8) and Imiquimod (TLR7) stimulation. Where 4National Microbiology cells from HESN had cellular activation in response to TLR7 and TLR7/8 stimulation. PBMCs from HESN Laboratories-Public women produced greater amounts of proinflammatory cytokines and type II Interferons in response to ssRNA TLR7/8 stimulation, but lower type I interferon, IL-10 and interferon induced protein-10 responses Health Agency of to Imiquimod TLR7, compared to those of NN. A balance between proinflammatory cytokines and anti- Canada, Winnipeg, MB inflammatory cytokines was observed in the cytokine environment established following TLR7 and TLR7/8 stimulation of PBMCs from HESN women. The PBMCs of HESN also had higher TLR7 and TLR8 receptor expression and activation TLR signaling pathway, with differences in activation of components of cytokine signaling pathways, compared to those of NN women. This study revealed differences in cellular activation, cytokine production, cytokine milieu, activation of TLR and cytokine signaling pathways between HESN and NN. These difference point to the existence of differences in innate function that may be related to protection against HIV. Low Dose Intradermal Infection with Trypanosoma congolense Leads to Expansion of Regulatory T cells and Enhanced Susceptibility to Reinfection Chukwunonso Onyilagha 1, Shiby Kuriakose 1, Rani Singh 1, Ifeoma Okwor 2, Jude Uzonna 1, 2

1Department of BALB/c mice are highly susceptible to experimental Trypanosoma congolense infection. Interestingly, a Immunology, Faculty of recent report showed that these mice are relatively resistant to primary intradermal low dose infection. Paradoxically, repeated low dose intradermal infections predispose to enhanced susceptibility to an Medicine, University of otherwise non-infectious dose challenge. Here, we explored the mechanisms responsible for this low- Manitoba, dose-induced susceptibility to subsequent low dose reinfection. We found that akin to intraperitoneal 2Department of infection, low dose intradermal infection leads to production of IL-10, IL-6, IL-12, TGF-β and IFN-γ by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low dose intradermal Medical Microbiology, infection led to expansion of CD4+CD25+Foxp3+ cells (Tregs) in both the spleens and lymph nodes draining Faculty of Medicine, the infection site. Depletion of Tregs by anti-CD25 mAb treatment during primary or before reinfection University of Manitoba, following repeated low dose infection completely abolished the low dose-induced enhanced susceptibility to rechallenge infection. In addition, Tregs depletion was associated with dramatic reduction in levels Winnipeg, MB of TGF-β and IL-10. Collectively, these findings show that low dose intradermal infection leads to rapid expansion of Tregs and these cells mediate enhanced susceptibility to subsequent infection. Investigating the role of p53 during Mammalian Reovirus infection of Transformed Host Cells Kolawole Opanubi, Jieyuan Jiang, Lou Wang and Kevin Coombs

Department of Medical Introduction: Mammalian Reoviruses (MRVs) are the prototypic members of the orthoreovirus genus in Microbiology and the family Reoviridae. This family represents the only group of dsRNA viruses that infect mammals. MRVs can infect and induce apoptosis in susceptible host cells; however, the precise molecular requirements and Infectious Diseases, signaling pathways of MRV-induced host cell apoptosis are not fully known. We are studying the role of the University of Manitoba, tumor suppressor protein, p53, in MRV-induced host cell apoptosis. p53 is a transcription factor involved in Winnipeg, MB determining the fate of cells stressed by viral infection or genomic damage, directing such cells towards cell cycle arrest or apoptosis. p53 intervenes at many points in apoptosis pathways and activates NF-κB in the signaling pathway to apoptotic cell death. This makes p53 a logical target for studying NF-κB dependent MRV-induced apoptosis. Methods: The human, p53 null, H1299 large cell carcinoma cell line and various p53 expressing cell lines were infected with MRV to study viral replication in the presence and absence of p53. Infectivity was monitored via plaque assay. The expression of p53, and p53 regulated genes in select cell lines was monitored by RNA protection assay. We are currently studying MRV growth and induced cell death in the p53 null H1299 cell line and H1299 cells stably transfected to express p53. Results We found that MRV replicates with similar efficiency in p53 null and p53 expressing cell lines. L929 cells were found to undergo apoptosis upon MRV infection while H1299 cells did not undergo cell death under similar infection conditions. RNA protection assay studies on the effect of MRV infection on p53 regulated host genes found proapoptotic Bax, and cell cycle arrest related p21 and GADD45α gene expression were increased 2-4 fold in L929 and H1299 cells after infection. MRV did not induce transcription of p53 mRNA suggesting expression of these aforementioned genes occurs in a p53 independent manner. Neurotransmitter Signaling via NMDA Receptor Enhances Production of a Potential Th2 Cell Survival Factor from CD4+ T Cells Kanami Orihara, Sonia Charran, Kent T HayGlass, Redwan Moqbel

Department of Background: Previously, we have shown that activated human CD4+ T cells express functional N-methyl- Immunology, Faculty of D-aspartate (NMDA) receptors (NMDA-R). NMDA-R stimulation induced selective cell death in Th1 cells, allowing Th2 cells to survive. NMDA-R is a type of glutamate receptors, overactivation of which can result in Medicine, University of various neurodegenerative conditions. Recently, several reports suggested that amphiregulin, an epithelial Manitoba, Winnipeg, growth factor (EGF) family member, may be associated with corticosteroid resistance in asthma. In this MB study, we aimed to investigate whether signaling via NMDA-R enhances amphiregulin expression and secretion from CD4+ T cells and thus contributes to NMDA-R-mediated differential cell survival in CD4+ T cell subsets. Methods: Human CD4+ T cells were isolated from PBMC from consenting adult healthy donors and activated with anti-CD3/CD28 antibodies. Expression levels of mRNA and protein were determined using quantitative real-time PCR and ELISA, respectively. Cell viability (metabolism) was detected by MTT assay. Results: Glutamate treatment in vitro significantly enhanced amphiregulin expression and production from activated human CD4+ T cells. Also, exogenous amphiregulin treatment promoted CD4+ T cells survival. This effect became obvious when cells were cultured in the presence of IL-4. Conclusion: We found amphiregulin production from human CD4+ T cells, which is enhanced following NMDA receptor activation. Evidence that exogenous IL-4 enhances amphiregulin production and effects implied an association of amphiregulin with Th2, but not with Th1 cells. Thus, we suggest that amphiregulin may be a potential survival factor for Th2 cells. Diabetes in Pregnancy Among First Nations Women in Alberta: A Multiphase Mixed Methods Approach Richard T. Oster and Ellen L. Toth

Department of Introduction: The purpose of this doctoral research was 1) to generate an epidemiological profile of First Medicine, Faculty of Nations diabetes in pregnancy in Alberta; and 2) to qualitatively explore among First Nations women both the experience of diabetes in pregnancy and the contributors to achieving a healthy pregnancy. Dentistry/Medicine, Methods: De-identified provincial administrative data of delivery records was obtained (years 2000-2009). University of Alberta, Pregestational, past obstetrical, and delivery outcomes and problems were compared by ethnicity and Edmonton, AB diabetes status. Rates and longitudinal changes of gestational diabetes (GO) and pregestational diabetes were compared by ethnicity. Diabetes in pregnancy predictors were explored via logistic regression. A focused ethnography was conducted with 12 First Nations women with previous diabetes in pregnancy that sought care in Edmonton. Unstructured interviews were carried out, recorded, transcribed, and subject to content analysis. Results: First Nations women were more likely to have antenatal risk factors and adverse infant outcomes, which was compounded by diabetes. Although GO rates were higher among First Nations women, prevalence grew significantly only in non-First Nations women. The longitudinal rates of pregestational diabetes were generally steady, yet First Nations women endure a nearly two-fold higher prevalence. Being of First Nations descent was an independent predictor of diabetes in pregnancy. The experience of diabetes in pregnancy was one wrought with difficulties but balanced to some degree by positive lifestyle changes. A struggle for control permeated the pregnancies but having a strong support system and the necessary resources allowed women to take control of their health. Conclusions: As high-risk pregnancies and poor outcomes are more common among First Nations women regardless of diabetes status, efforts must be made to improve pregnancy care. Specifically, these efforts should strive to enhance the support systems of these women, increase their sense of autonomy, and raise awareness of diabetes in pregnancy and its accompanying challenges. Transcription Factors NF-κB, CREB and Egr-2 and their Potential Role in Long Term Memory Payam S. Pahlavan, Eric Platt, Shoug Alashmali, Chris Cadonic, Melanie Neuendorff, Miyoung Suh, Wanda Snow and Benedict C. Albensi

Department of Objective: Long term memory formation is associated with the activation of transcription factors (TF). TFs Pharmacology & believed to be involved in long term memory include the cAMP-response element binding protein (CREB), nuclear factor kappa B (NF-kB) and early growth response-2 (Egr-2) protein, which likely play different roles Therapeutics, in short vs. long term memory and in memory encoding, consolidation, and/or reconsolidation stages. We University of Manitoba, hypothesized there would be a difference among these TFs in expression and activation levels in pre- versus Winnipeg, MB post-learning phases of the Morris water maze (MWM), a hippocampal dependent memory task. Methods: CD1 and C57BL/6 mice (1-2 mos old) were assigned into one of three experimental groups or a control group (exposed to maze with no training). Mice were trained in the acquisition phase for 5 d (hidden platform paradigm). The platform was removed for the probe phase (on day 6 or 12). Search strategies, escape latency, time spent in target quadrant, swim speed, and the number of passes over the missing platform, were measured during these 2 phases. Mice were killed ~30 mins or 24 hrs after the probe test. Hippocampi were isolated and Western blots, IHC, ELISAs, and EMSAs were conducted to determine the activation and expression levels of CREB, p-CREB, NF-kB subunits (IkB, p105, p65, p50), and Egr isoforms (1-4), pre- versus post- MWM training. Results: In CD1 mice, escape latency was decreased over 5 d indicating animals learned the location of the platform; also, the NF-kB p50 subunit and CREB expressed significantly higher in the control group vs. the MWM trained mice (p = 0.02 & p = 0.0009, respectively) killed on day 6. In a study with C57BL/6 mice, mice learned the location of the platform (p < 0.05) over 5 d. In IHC assays following MWM training, NF-kB p65 subunits and p-CREB expressed at higher levels in mice killed at 24 hrs following the probe test on day 6, than in the other groups. Conclusion: As a result of MWM training, differences exist in the expression and/or activation of CREB, NF- kB, and Egr-2. Polycystic Ovary Syndrome: An Opportunity to Reduce the Incidence and Burden of Ischemic Heart Disease Among Young Women? Kristi Panchuk1, Martha Mackay1, Karin Humphries2, Anthony Cheung2, Priscilla Taipale1

1School of Nursing, Introduction: Over the past 40 years, the magnitude of improvements in cardiovascular (CV) mortality has University of British been greater for men than for women. Most concerning is the high rate of mortality in young women who experience ischemic heart disease (IHD). Since IHD is largely preventable, it is imperative that researchers 2 Columbia, Faculty of identify the age- and sex-specific conditions that contribute to IHD. One sex-specific condition that may Medicine, University of contribute to the disease burden of IHD is polycystic ovary syndrome (PCOS), which affects between British Columbia, 7% and 18% of women of reproductive age. Pathognomonic features of PCOS include anovulation, hyperandrogenism, and/or polycystic ovaries on ultrasound assessment; dyslipidemia, insulin resistance and Vancouver, BC obesity, known risk factors for IHD, are also common. Methods: This literature review examines studies from gynaecology, endocrinology, and cardiology, and focuses on a) meta-analyses of CV risk factors in PCOS and b) studies examining the relationship between PCOS and CV outcomes such as multi-vessel coronary artery disease and CV event-free survival. Results: Studies consistently demonstrate that PCOS worsens women’s risk profiles for CV disease, particularly when risk factors can persist beyond menopause. Recent meta-analyses of mechanistic studies have suggested that PCOS increases CV risks through inflammatory, endothelial, and coagulation alterations. However, it is unclear if PCOS contributes to IHD among younger women. Furthermore, the presence of these intermediate risk factors does not necessarily translate into poor CV outcomes. Conclusion: This literature review outlines how current evidence about the effect of PCOS on CV outcomes is inconclusive because of the challenges associated with diagnosing PCOS during and after midlife, the confounding effect of obesity on the manifestations and sequelae of PCOS, and the relatively low incidence of IHD among women under 65 years of age. Future research to examine CV outcomes in women under 65 with PCOS will create opportunities for tailored or targeted primary and secondary prevention strategies. Host Cell Proteome Changes Induced by the Hepatitis C Virus NS3/4A Protein. Aileen Patterson, Xiaojie Hu, Stuart McCorrister, Garret Westmacott, and Michael Carpenter

Department of Medical Hepatitis C virus (HCV) infects an estimated 200,000 people here in Canada, and is the leading cause of Microbiology, liver transplants in North America. If left untreated, chronic infection can lead to liver fibrosis, steatosis and hepatocellular carcinoma (HCC). One of the HCV protein complexes, made up of the non-structural proteins University of Manitoba, 3 and 4A (NS3/4A), is thought to attribute to HCC development. in vitro, the NS3 protease has been shown Winnipeg, MB, Public to induce advanced cellular transformation, and form tumours in nude mice. However, the mechanism by Health Agency of which this occurs is not known. The objective of this study was to evaluate the overall effect of NS3/4A to the host cell proteome, and to determine if any of these protein abundance changes account for the Canada, Winnipeg, formation of HCC. To examine these questions, I constructed cell lines stably expressing either NS3/4A or MB GFP. Abundance changes to the host cell proteome upon NS3/4A expression were evaluated by quantitative proteomics using SILAC (stable isotope labeling of amino acids in cell culture) in quadruplicate with reverse labeling. In total, over 6000 proteins were identified (>70,000 peptides), 23 of which were differentially regulated by NS3/4A expression, including Prostaglandin Reductase 1 and Neurofilament 4. Further characterization of these and other proteins showing significantly altered abundances is ongoing. A Novel Phosphorylation Site in SHIP Mediates Binding to Nck and Regulates Actin Polymerization in B cells Samantha D. Pauls1, Arnab Ray2, Mark S. Cragg3, Aaron J. Marshall1,2

1Department of Leukocyte activity is controlled by balancing activating and inhibitory signals in order to generate effective Biochemistry and immune responses while avoiding tissue damage and autoimmunity. This project examines an important regulatory circuit in B cells that involves the lipid phosphatase SHIP, a regulator of the phosphoinositol Medical Genetics, 3-kinase signaling pathway. SHIP is known to dampen cellular responses downstream of B cell receptor 2Department of ligation, classically when the inhibitory receptor FcγRIIB is co-ligated. This large protein contains multiple Immunology, protein-protein interaction domains. Accordingly, various phosphatase-independent adaptor functions have been described. Prediction tools suggest that SHIP might interact with the adaptor protein Nck by University of Manitoba, way of an uncharacterized phospho-tyrosine site in its C-terminus. Here, we aim to confirm this interaction Winnipeg, MB, 3Cancer and, independently, resolve the contribution of the phospho-site to the overall function of SHIP. We have Sciences Unit, demonstrated binding by surface plasmon resonance analysis as well as protein pulldowns. We also performed a series of preliminary experiments where a vector control, wild type SHIP or mutant SHIP lacking University of the tyrosine residue of interest is over-expressed in a human B cell line for functional comparison. We used Southampton, phalloidin staining to measure F-actin content and fluorescence recovery after photobleaching experiments Southampton, UK to measure the turnover rate of fluorescently tagged monomers within actin filaments. Cell spreading responses to immobilized B cell receptor ligand were also compared. Finally, differences in recruitment to the plasma membrane upon stimulation via the B cell receptor alone or co-ligated with FcγRIIB were examined by confocal microscopy. Our study describes for the first time an interaction between the lipid phosphatase SHIP and the adaptor protein Nck and explores the function of a novel phospho-site in SHIP that may mediate this interaction. A Rat Model of High Fat and Sucrose Diet-Induced Diabetes During Pregnancy Troy J. Pereira, Mario Fonseca, Xuili Ma, Vern W. Dolinsky

Manitoba Institute of Hypothesis: We hypothesize that the consumption of HFS by pregnant rats results in hyperglycemia, Child Health (MICH), hyperinsulinemia and glucose intolerance that is characteristic of GDM. Furthermore, we hypothesize that rat offspring born from HFS dams will be more susceptible to metabolic disorders than offspring from Winnipeg, MB control dams. Methods: To induce GDM, female rats consumed HFS (45% fat) diets during pregnancy, while control dams were fed low (10%) fat diets. Maternal body weight, food consumption and blood glucose measurements were recorded. One-day old offspring from control and GDM dams were analyzed for their body and tissue weights. Results: During pregnancy, HFS dams weighed 18% more than control dams. Furthermore, HFS fed dams were glucose intolerant and hyperglycemic compared to controls. There were 39% fewer pups from HFS damns, though the pups from HFS dams were 11% larger than the pups from the control dams. Preliminary data also suggests that offspring from HFS dams are more glucose intolerant than the control dams. Conclusion: We conclude that in rats, the consumption of HFS diets by pregnant dams leads to the development of hyperglycemia, hyperinsulinemia and glucose intolerance, characteristic of GDM. These maternal factors may also contribute to adverse effects during fetal development. MicroRNA 200b in Branching Morphogenesis of Normal and Abnormal Lung Development – Congenital Diaphragmatic Hernia Patricia Pereira Terra1,2,4,5, Anouk Chantal Deden1,2,7, Fuqin Zhu1,2, Barb Iwasiow1,2, Jorge Correia Pinto4,5,6, Richard Keijzer1,2,3

1Manitoba Institute of Background: Abnormal lung development in congenital diaphragmatic hernia (CDH) still remains poorly Child Health, Winnipeg, understood and is directly correlated with a high morbidity and mortality in CDH patients. MB, 2Department of MicroRNAs (miRNAs) are a class of post-transcriptional regulators that revolutionized our view of gene Surgery, 3Department expression regulation. They have important roles in cellular and developmental processes [1]. of Physiology, In our previous work, we showed that mir 200b expression is affected by CDH. University of Manitoba, The aim of this study was to evaluate the role of mir200b in branching morphogenesis in a lung explant Winnipeg, MB, 4Life and model using lungs from normal and CDH rats. Methods: At 13.5 days post-conception, we assigned rat fetuses 4 experimental groups for lung explant Health Sciences culture: control, control plus mir 200b inhibitor, nitrofen, and nitrofen plus mir200b mimic. Morphometric Research Institute analysis of cultured lungs was performed and Real Time PCR in order to quantify the expression levels of (ICVS), School of Health mir200b in the lungs. Sciences, University of Results: When compared with control lungs, groups treated with inhibitors showed a decrease in branching, Minho, Braga, Portugal, perimeter and area. Nitrofen plus mimics groups when compared with nitrofen lungs, showed an increase in 5 ICVS/3B’s - PT the same parameters. Government Associate Regarding mir 200b expression, CDH lungs showed less expression of mir200b than normal lungs. Nitrofen Laboratory, Braga/ treated lungs with mimics showed higher levels of mir200b than nitrofen lungs and control lungs with Guimarães, Portugal, inhibitors showed lower expression when compared to the control lungs. 6Department of Conclusion: This study reinforces the idea that Mir 200b is involved in lung development and shows, for the Pediatric Surgery, first time, that the administration of mir200b mimics and inhibitors increases or decreases respectively lung Hospital de Braga, growth and branching. Braga, Portugal, These findings strongly suggest that microRNAs could be used as potential therapeutical treatments in CDH 7Erasmus University patients. Rotterdam, Rotterdam, Netherlands A Preliminary Study of Infant Nasal Microbiome Diversity Applying cpn60 Universal Target (UT) as a Phylogenetic Marker S. Peterson1, N. Knox2, G. Golding1,2, J. Embree1, F. Fleming1, S. Fanella1, M. Graham1,2, G. Van Domselaar1,2, M.R. Mulvey1,2

1Department of Objective: A protocol was developed to study the nasal microbiome applying an internal cpn60 universal Medical Microbiology, target (UT) amplicon as a bacterial phylotyping marker using next generation sequencing. The UT can be used to identify and differentiate organisms in metagenomic samples to the species level. We aimed to University of Manitoba, determine the typical composition of anterior nares microbiota for developing infants over time, and to Winnipeg, MB, explore diversity of the infant nasal microflora and the relation to microflora observed within their primary 2National Microbiology caregivers. Methods: Ethics approval was obtained from the U of Manitoba and PHAC. Nares swabs were collected Laboratory, Public from 40 two-week old infants and their primary caregivers over a 1-year period using a single swab in one Health Agency of nare for each subject. These samples were treated with the MoLysis kit (Molzym) to diminish contaminating Canada, Winnipeg, MB human DNA, and bacterial template DNA was augmented using the GenomiPhi (GE Healthcare) whole- genome linear augmentation procedure. The cpn60 UT target was PCR amplified using degenerate primers and sequenced using high throughput 454/GS FLX pyrosequencing. Reads were quality-filtered and matched to the cpn60 database using an 80% nucleotide identity cutoff. Results: Reads were assigned to 23 unique phylotypes, with the 12 most abundant phylotypes accounting for >98% of the community. The remaining inferred phylotypes represent rare taxa. Infant/caregiver samples contained 63%/76.5% Actinobacteria, 35%/18% Firmicutes and 2%/5.5% Proteobacteria. Trace amounts of Bacteroidetes were found in both groups. 17% of infant reads were streptococci, compared to 3.9% in caregivers. Lactobacilli represented 0.04%/0.003% (infants/caregivers) and Actinobacteria represented 4%/17%. Conclusions: Lactobacilli and streptococci were more commonly found in infants, while Actinobacteria were more likely to be associated with caregivers. This data is in agreement with studies of healthy adult human nares microflora previously described in the literature, demonstrating that our sampling and template processing protocols are effective in identifying organisms within the anterior nares. Perinatal Antibiotic Exposure of Neonates and Mothers in Manitoba Hospitals R.P. Persaud1, M.B. Azad2, A.B. Becker3, A.L. Kozyrskyj2, and the CHILD Study Investigators4

1Department of Background: Allergy and asthma are a significant burden on individuals and the healthcare system. Changes Pharmacy, University to the composition of an infant’s microbiota may increase the risk of atopic diseases and asthma. Infants exposed to antibiotics, particularly broad-spectrum antibiotics, have an increased risk of asthma. More of Manitoba, research is required to determine the relationship between changes to the microbiota due to antibiotic 2Department of exposure and the risk of atopy and asthma. Our research aimed firstly to describe the range of perinatal Pediatrics, University of antibiotic exposures in Manitoba-born infants and the indications for antibiotic treatment; secondly to determine if an association exists between maternal health status and common indications for perinatal Alberta, Edmonton, AB, antibiotic treatments in Manitoban hospitals. 3Pediatrics and Child Methods: Data was gathered by retrospective chart review of 280 mother-neonate pairs enrolled in the Health, University of Canadian Healthy Infant Longitudinal Development (CHILD) study. Deliveries occurred at three Manitoban hospitals (St. Boniface, Health Sciences Centre, and Boundary Trails Health Centre). Antibiotic exposures of the Manitoba, Winnipeg, mother and neonate, and measures of maternal health status were systematically recorded for statistical 4 MB, Canadian Healthy analysis by chi-squared test and Fisher’s exact test. Infant Longitudinal Results: In total, 110 neonates (39%) were exposed to antibiotics in hospital. Of these, 98 (35%) were exposed Development Study, indirectly via the mother, while 12 (4%) were both directly and indirectly exposed. Penicillin G and the relatively broader-spectrum cefazolin were the two most commonly administered antibiotics. The most common Winnipeg, MB indications for mothers to receive antibiotics included Cesarean-section (21%), Group B Streptococcus (22%), and rupture of membranes greater than 18 hours before birth (15%). Obese mothers required Caesarean- section at a significantly greater rate than women of a normal and above-average weight (p=0.05). Hyperten- sive mothers were positive for Group B Streptococcus at a significantly greater rate than non-hypertensive mothers (p=0.05). Conclusion: The majority of antibiotic exposures to Manitoban neonates in-hospital during the perinatal period occurred indirectly via the mother. The mother’s health status contributes to the chance of neonatal antibiotic exposure. Going forwards, the presented data will be used to determine the effect of antibiotic exposures on the fecal microbiota composition and allergic disease outcomes that have been established by the CHILD study. A Single, Ultra High Dose Aminoglycoside Therapy in A Rat Model of E. coli Induced Septic Shock Amarnath Pisipati1, Ehsan K. Esfahani3, Brian Blakely2, Robert Ariano3, Anand Kumar1

Department of Medical Introduction: Septic shock is a life threatening condition often due to Gram negative bacterial infections Microbiology, and is responsible for around 200,000 deaths every year in United States of America alone. Our research focuses on determining the potential of a single, ultrahigh dose aminoglycoside therapy for accelerated Department of bacterial clearance with minimal to manageable side effects in a rat model of E. coli induced septic shock. Otolaryngology and Methods: Study in uninfected animals: Gentamicin was administered intravenously at 80 and 160 mg/kg Department of doses to anesthetized and intubated Sprague Dawley rats. Mean arterial pressure, heart rate, respiratory rate and percentage oxygen saturation were determined. The occurrence and time course of neuromuscular Pharmacology, paralysis/ventilator dependency, renal injury and auditory toxicity after ultra-high dosing of gentamicin University of Manitoba, was determined. Renal injury was determined by Creatinine clearance and histology. Auditory brain stem Winnipeg, MB response studies were performed to assess the auditory toxicity. Septic shock induced animals: Septic shock was induced in rats by surgical, intra-peritoneal implantation of a gelatin capsule containing E. coli bort (ATCC 700973) in a fibrinogen and alfa-cellulose matrix. Gentamicin was intravenously administered at 5, 10, 20, 40, 80 and 160 mg/kg doses. Results: Ultra high gentamicin dosed animals were found to develop neuromuscular paralysis with ventilator dependency for 20 – 40 minutes and recovered thereafter. Rats did not exhibit any auditory and renal toxicity. Complete eradication of bacterial counts from blood occurred following ultra-high doses. Conclusion: Rats were able to tolerate single, ultra-high doses of gentamicin with complete eradication of bacterial counts from blood. Neutrophil Chemotactic Decision Making at Sites of Sterile Injury Keir Pittman, 1,2, Paul Kubes1,2

1Department of Introduction: Neutrophils migrate towards sites of injury and infection and are essential effectors of the Physiology & acute inflammatory response. In instances of sterile injury wherein no microbial pathogens are present, neutrophils will migrate towards sites of sterile injury following first intermediate level directional cues Pharmacology and generated from the body’s own cells, and then formyl peptide receptor 1 signal into the precise focus of Medicine, Immunology injury. Using in vitro chemotaxis assays, we model neutrophil directional “decision making” in competing Research Group, gradients of chemokine and sterile injury signals Methods: Human neutrophils were isolated from peripheral blood and placed in in vitro under agarose University of Calgary, chemotaxis assays. Chemotactic behavior was observed in presence of necrotic HEK293 tissue culture 2Snyder Institute for cells disrupted using a thermal cautery probe. Behavior of neutrophils between competing gradients of Chronic Diseases, interleukin-8 or fMLP (at their respective optimal concentrations) and sterile injured necrotic tissues was observed. University of Calgary, Results: In the presence of necrotic cells, movement of neutrophils towards interleukin-8 is effectively Calgary, AB reduced. However, the presence of damaged cells does not inhibit chemotaxis towards bacterial chemoattractants, even when the latter are present at suboptimal concentrations. This indicates that signals from sites of sterile injury are hierarchically equivalent to bacterial “end-target” ligands. Conclusion: Formyl peptide receptor 1 signals originating from sterile injured tissue culture cells, likely formyl peptides released from damaged mitochondria, play a role in directing neutrophils into a focus of necrotic cells. Neutrophils will therefore ignore competing gradients of chemokine in favor or “end-target” sterile injury cues. Movement towards competing gradients of synthetic bacterial ligands is unaffected. This behavior may preserve surrounding viable tissues from “collateral damage” caused by the recruitment of neutrophils to sites if sterile injury. Understanding the behavior of neutrophils in the acute response to sterile injury lays important ground work for identifying potential therapeutic targets that could limit detrimental inflammation at sites of injury without compromising the body’s response to pathogen. Innate Defence Regulator (IDR) Peptides in Airway Inflammation and Remodeling Hadeesha Piyadasa, Leola N.Y. Chow, Sujata Basu, Jackie Schwartz, Andrew Halayko and Neeloffer Mookherjee

Manitoba Centre for Introduction: An essential function of innate immunity is to initiate inflammatory responses required for Proteomics and resolution of infections and neoplasms. Breakdown in the regulation of inflammation leads to sustained elevated inflammation associated with the pathologic consequences observed in chronic inflammatory Systems Biology, diseases e.g. asthma, inflammatory bowel disease, and arthritis. A group of natural immune-modulatory Department of Internal molecules known as cationic host defence peptides (HDP) and their synthetic derivatives known as Medicine, Department innate defence regulator (IDR) peptides, contribute to resolution of infections and pathogen-induced inflammation. The objective of this study was to examine the effect of IDR peptides in Th2-mediated of Immunology, inflammation and lung remodeling in allergic asthma. Department of Method: House dust mite (HDM)-challenged murine model of allergic asthma was used to study the effects Physiology, University of a 12-amino acid IDR peptide. Lung mechanics was evaluated using flexiVent small animal ventilator. ELISA and flow cytometry was used to monitor cytokine production in serum, lung extracts and bronchoalveolar of Manitoba, Winnipeg, lavage fluid (BALF). Western blots were used to monitor protein expression in lung epithelial cells. MB Results: We showed that IDR peptide do not induce any pro-inflammatory cytokines such as TNF, IL-1, IL-4 or IL-13, either in BALF, serum or lung tissue extracts in the murine model. The 12-amino acid IDR peptide reduced HDM-induced Th2-cytokine IL-13 in the lung tissues extracts. Monitoring lung mechanics, we showed that HDM-challenged mice had increased airway and tissue resistance to inhaled methacholine compared to naïve mice, whereas administration of the IDR peptide significantly reduced these responses. To elucidate the mechanism of this process, we monitored the TGFB-1-induced epithelial-to-mesenchymal transition (EMT) in human bronchial epithelial cells, and showed that the IDR peptide suppressed TGFB-1- induced expression of vimentin, a protein marker of EMT. Conclusion: IDR peptide significantly improved lung mechanics of HDM induced asthmatic mice and also reduced production of the Th2-cytokine IL-13 in the lung tissues. The IDR peptide suppressed TGFB-1 induced expression of vimentin, suggesting that the peptide can interfere in EMT. This study demonstrates that IDR peptides have the potential to control Th2-induced chronic inflammation and lung remodeling in allergic asthma. Using Molecular Profiling to Characterize Human Embryonic Stem Cell-Derived Hormone-Positive and Hormone-Negative Cells Kacey J. Prentice, Christina L. Basford, Emma M. Allister, M. Cristina Nostro, Gordon Keller, and Michael B. Wheeler

Department of Objective: It is possible to generate islet hormone expressing cells from hESCs in vitro; however, it has been Physiology, University demonstrated that these hormone-expressing cells mature only into α cells when transplanted, and that only a subpopulation of the non-hormone expressing pancreatic endoderm fraction produce functional β of Toronto, Toronto, cells in vivo (Kelly et al., 2011). In this study we aim to 1) characterize hormone-expressing hESC-derived cells ON to determine why these cells adopt and α cell fate in vivo, and 2) characterize the hormone-negative hESC- derived cells to define the composition of this population. Methods: hESC-derived endocrine-like cells were differentiated in vitro using the Nostro protocol from a genetically modified HES3 line expressing GFP under the insulin promoter. FACS was used to isolate purified populations of insulin-positive (Ins+) or insulin-negative (Ins-) cells to be used for all experiments. QPCR established that our Ins- population was in fact hormone-negative. In all cases cells were compared to adult human islets. We performed molecular characterization using microarray and LC/MS/MS to identify differentially expressed genes and proteins. To validate our findings, we performed functional characterization including immunohistochemistry, secretion assays, electron microscopy, and electrophysiology. Results/Conclusions: Our Ins+ cells display many α cell characteristics in vitro including glucose-stimulated glucagon secretion and a deficiency of functional L-type calcium channels. Glucagon was among the top five most abundantly expressed proteins, andα cell-specific transcription factors Arx, Brn4, and Irx1/2 had significantly higher expression in Ins+ cells compared to human islets by microarray. Whileβ cell proteins were present, microarray analysis revealed a significant deficiency inβ cell-specific transcription factors Pax4 and Nkx6.1. Therefore, the Ins+ population is likely adopting an α cell fate in vivo due to high expression of α cell-specific factors, which have already committed these cells to anα cell lineage in vitro. When examining the Ins- fraction, microarray analysis revealed relatively strong expression of pancreatic endoderm markers including Pax6 and NeuroD. Intriguingly, these cells had extremely high expression of liver- and neuronal-specific genes including the apolipoproteins and alphafetoprotein from the liver and β3 tubulin and neuropilins from neurons. Markers of other non-pancreatic foregut derivatives including duodenum and lung were not significantly expressed. Interestingly, when culturedin vitro, the Ins- fraction takes on a neuronal phenotype, suggesting that this is the dominant feature in an extremely heterogeneous population. Expression of genes from the pancreatic lineage suggests that a minority of cells within this population may be true β cell progenitors, however, efforts need to be made to determine a way to isolate this population in vitro before they represent a viable transplant option. Ex Vivo Expanded Human Umbilical Cord Blood Myeloid Progenitor Cells Stimulate Vascular Regeneration David Putman and David Hess

Department of Effective revascularization therapies are critical to the treatment of ischemic diseases including myocardial Physiology and infarction, stroke and peripheral vascular disease. We have recently reported that human UCB-derived multipotent myeloid progenitor cells, prospectively purified based on high aldehyde dehydrogenase Pharmacology, Western activity (ALDHhi), can promote the recovery of perfusion and vascularization in immunodeficient mice with University, London, acute unilateral hindlimb ischemia. Unfortunately, the rarity of ALDHhi progenitor cells (0.4±0.1% of total ON mononuclear cells) impedes clinical implementation of cell-based therapies due to difficulty achieving clinically relevant cell numbers. To address this issue, we have optimized ex vivo culture protocols to increase the number of UCB ALDHhi cells available for therapy. UCB ALDHhi cells were grown on fibronectin-coated plates in serum-free, X-vivo15 media supplemented with recombinant human SCF, Flt-3L, and TPO (10ng/ mL). After 6 days of culture, we observed 14.0±5.6 fold expansion in total cell number (n=10), and 13.2±2.7% of cells retained high ALDH-activity, Expanded ALDHhi cells maintained primitive progenitor (77.6±4.2% CD34+, 48.7±7.7% CD133+, 5.46±1.2% CD117+) and early myeloid markers (99.9±0.2%, CD33+), an expression pattern similar to freshly isolated UCB ALDHhi cells (n=5). Furthermore, expanded cells maintained multipotent myeloid colony forming cell function in methylcellulose media (1 CFU in 10 cells). Remarkably, intramuscular injection of these expanded myeloid progeny into mice with acute unilateral ischemic injury stimulated robust vascular recovery with kinetics similar to uncultured UCB ALDHhi cells. Within 7 days post-transplantation, ex vivo expanded myeloid progenitors (5x105 cells) significantly accelerated the recovery of perfusion (RP) in the ischemic limb (RP=40±7%, n=8) compared to saline-injected mice (RP=25±8%, n=15, p<0.05), and augmented perfusion was maintained for one month post-transplantation (RP=71±16% vs 39±12%, p<0.01). Progenitor cell transplanted mice also showed higher vWF+/CD31+ capillary density in the ischemic limb compared to saline injected controls (p<0.01). Collectively, these data indicate that UCB ALDHhi cells can be efficiently expanded in serum-free ex vivo culture without significant loss of progenitor phenotype or vascular regenerative capacity, and provide proof-of-concept data to support the clinical translation of allogeneic cell- based therapies for ischemic disease. Structure – Function Analysis of the Ligand Binding Pocket of Bitter Taste Receptor T2R4 Sai Prasad Pydi1, Ken Nelson2, Michele Loewen2, Rajinder P. Bhullar1 and Prashen Chelikani1

1Department of Oral Bitter taste receptors (T2Rs) belong to the G protein coupled receptors super family. The 25 human T2Rs are Biology, University of activated by wide range of bitter compounds which includes alkaloids, terpenoids, peptides and amines. The human T2R4 is activated by around 16 bitter agonists, which are mostly sesquiterpene lactones, amine Manitoba, Winnipeg, derivatives, purine analogs and aromatic quinolines. From molecular modeling analysis we found that, the MB plant hormone abscisic acid (ABA), an ABA biosynthetic precursor and few of the ABA metabolites docked with high affinity to T2R4. The ability of these compounds to activate or inactivate T2R4 was analyzed by calcium imaging in a HEK293 cell based assay system. We discuss the results in the context of T2R4 structure and function. Characterizing the Importance of the HIV Integrase Mutation G118R in Subtypes C and CRF02_A/G Peter K. Quashie, Thibault Mesplède, Tamar Veres, Said Hassounah, Yingshan Han and Mark A. Wainberg

McGill University AIDS Background: The HIV-1 subtypes B, C and CRF02_A/G account for greater than 75% of all infection and Center, Lady Davis it is important to understand the variations of viral control in these different subtypes. HIV integrase (IN) mediates the insertion of viral DNA into host DNA, a process known as integration. IN was an early choice for Institute-Jewish anti-HIV drug development, leading to the clinical licensure of the integrase inhibitors (INIs), raltegravir (RAL) General Hospital, in 2007 and Elvitegravir (EVG) in 2012; a third, dolutegravir (DTG) is expected to be approved for clinical use Montreal, QC in 2013. RAL and EVG have a low genetic barriers for resistance development with extensive cross resistance; DTG has a high genetic barrier and has not selected resistance mutations in INI-naive patients. The IN drug resistance mutation G118R was reported in a CRF02_A/G HIV-1 infection and conferred resistance to RAL. We selected G118R in combination with E138K in cell culture selection experiments with the IN strand transfer inhibitor (INI) MK-2048; DTG also selected G118R in CRF02_A/G and subtype-C viruses, followed by H51Y. Our objective was to understand the mechanisms by which G118R ± H51Y or E138K differentially affect integration and its inhibition in the three subtypes studied. Methods: Strand-transfer and 3’ processing activities as well as INSTI inhibitory constants were measured with purified recombinant HIV-1 subtype AG, B and C enzymes harboring the G118K ± H51Y or E138K mutations using microtiter plate assays. Drug dose-dependent anti-viral phenotypic assays were performed with infectious HIV virus containing either wild-type IN or mutant IN. Modeling of HIV-1 IN intasomes was performed using the programs I-TASSER, PyMol, UCSF Chimera and YASARA based on the published drug- bound structure of prototype foamy virus integrase. Results: The effects of G118R ± H51Y or E138K on IN activity and susceptibility to DTG, RAL and EVG differs based on subtype. Individual H51Y and E138K in isolation did not have a deleterious effect on IN activity in any subtype, despite the combination of H51Y/G118R being deleterious to activity in all three subtypes. Resistance to DTG of G118R was in the order CRF02_A/G> subtype B> subtype C and resistance was increased in the presence of H51Y or E138K. In CRF02_A/G integrase the presence of G118R in isolation is sufficient to cause greater than 15 FC in resistance to RAL. Conclusion: In the context of ongoing RAL or DTG therapy, G118R may be clinically relevant in subtype C and CRF02_A/G HIV infection. The combination of H51Y/G118R may be beneficial for the patients. Elucidating the 3D Structure of the Ebola Virus Nucleocapsid Melissa Rabb1,2, Daniel Beniac1, Shannon Hiebert1, Lindsey Lamboo1,3, Timothy Booth1,2

1National Microbiology Ebola is a `hot’ virus. Its extreme lethality, lack of treatments or vaccines, and its potential as a bioterrorism Laboratory, Public agent have made it a popular fictional topic and a significant public health concern. Ebola is a filamentous virus that can contain more than one genome, increasing the possibility of infection. Each Ebola virus Health Agency of genome is protected by a protein shell, called a nucleocapsid. At least three different viral proteins Canada, Winnipeg, MB, including the nucleoprotein, VP24, and VP35 self-assemble into a helical structure to form the Ebola 2Department of virus nucleocapsid; however, the arrangement of viral proteins within this structure is still unclear. We hypothesize that the nucleocapsid structure consists of an inner and outer helix. The inner helix is produced Medical Microbiology, by interactions between nucleoproteins and the negative sense RNA genome of Ebola, while VP24 and University of Manitoba, VP35 form the outer helix. In addition, our data suggests that VP24 and VP35 interact to form a protruding, 3Department of bridge-like structure on the exterior of the outer helix. To investigate our hypotheses, plasmids encoding the Ebola virus nucleocapsid proteins will be transfected into cells to generate nucleocapsid-like structures. Microbiology, Three-dimensional models of these nucleocapsid-like structures will be generated using cryo-electron University of Manitoba, microscopy and computer analysis. Comparisons of the nucleocapsid-like structures and Ebola virus native Winnipeg, MB nucleocapsids will be performed and protein arrangements within these structures will be determined. Unraveling the structure of the Ebola virus nucleocapsid will provide crucial knowledge for the design of antiviral drugs against this deadly virus. Inhibition of Disease Severity in DSS Model of Colitis by Intrarectal Administration of Cateslytin, a Chromogranin A Derived Peptide Mohammad Fazle Alam Rabbi1, Hong Ji1, Metz-Boutigues2 and Jean-Eric Ghia1

1Department of Aims: Chromogranin (Cg) A of the granin family of proteins is stored in enterochromaffin granules and Immunology, is established as a neuroendocrine tumor marker. CgA or its shorter bioactive fragments are increased in inflammatory bowel disease patients. However, very little is known about their role in gastrointestinal University of Manitoba, physiopathology. The aim of the present work was to test the involvement of cateslytin (CTL; CgA 344-358), a Winnipeg, MB, 2INSERM CgA derived peptide in colonic inflammation. U.997, Strasbourg, Methods: Mice received 5% dextran sulfate sodium (DSS) in drinking water for 5 days to induce colitis. CTL fragment was given (intrarectally, 0.5mg/kg) starting one day before induction of colitis and control France mice received vehicle. Disease activity index (DAI) was evaluated daily after induction of colitis. At sacrifice, disease severity and inflammation were evaluated clinically and myeloperoxidase activity (MPO) and C-reactive protein (CRP) was determined. In a second experiment, peritoneal macrophages were isolated from non-colitic C57BL/6 mice and pretreated with CTL for one hour and stimulated with LPS (100ng/ml) for 24 hours and cytokines were assessed. Macrophages were also treated with rhodamine-labeled CTL to assess peptide penetration. Results: CTL treatment reduced the onset of clinical disease as assessed by loose stools, weight loss and colonic bleeding.On day 4, 62.5% and 37.5% of CTL-DSS-treated mice had active bleeding and a weight loss over 5%, respectively compared to 83.33% and 66.66% of DSS-treated mice. 50% of DSS-treated mice had loose stool compared to 0% of CTL-DSS-treated mice. Composite score was significantly different for the last two days. Macroscopic scores and CRP level were 40% and 11% lower in CTL-DSS-treated mice compared to DSS-treated mice (p<0.05). Macrophages isolated from non-colitic mice showed a selective decrease of INF-g and IL-1b (-81% and 66% respectively) release when pretreated with CTL and stimulated with LPS. We also demonstrated that the rhodamine-labeled peptide penetrate macrophages. Conclusions: These results support the hypothesis that chromogranin-derived peptides modulate intestinal inflammation in a murine model of colitis. Identification of the molecular mechanism underlying the protective role of this peptide may lead to a novel therapeutic option in IBD instead of immune modulator drugs currently available. Identification of Host Cellular Factors that Interact with Influenza A Non-Structural Protein (NS1) Protein for Replication Md Niaz Rahim1,2, Patricia J. Sauder2, Peyman Ezzati2, Kevin M. Coombs1,2,3

1Department of Introduction: Influenza A viral (IAV) NS1 is a multifunctional protein and essential for viral replication Medical Microbiology, in host cells. It plays important role during IAV infection by suppressing the antiviral responses and inhibiting interferon (IFN) induction. The mechanisms of IAV-host interactions during infection and disease Faculty of Medicine, progression are yet to be discovered. Although some studies found few NS1 interacting host factor using University of Manitoba, recombinant IAV, my goal is to identify host factors that interact with native IAV NS1 and mechanistically Winnipeg, MB, determine how some of them affect different IAV growth. Methods: To detect native-NS1 interacting host factors we generated monoclonal antibodies (mAb) to NS1 2Manitoba Centre for of different types of IAVs by immunizing BALB/c female mice with His6-NS1 of H3N2 type and screening Proteomics and the hybridomas using GST-NS1 of H1N1 type. mAbs were isotyped and characterized by immunoblot, Systems Biology, slot blot, immunofluorescence microscopy after infecting canine MDCK, human A549 and mouse M1 cells. Linear epitope mapping of these mAbs were done. NS1 interacting proteins will be pulled down by Winnipeg, MB, immunoprecipitation, products will be identified by Mass Spectrometry (MS). 3Manitoba Institute of Results: 9 different broadly cross reactive mAbs were generated that can detect mostly native and Child Health, Winnipeg, denatured form of NS1 of six different IAVs (H1N1 and H3N2). Five different epitopes were also found and MB these can detect subcellular localization of NS1. A549 cells are infected with different IAVs. At early and late stages of infection both cytosolic and nuclear fractions will be subjected to perform immunoprecipitation using a mixture of NS1-mAbs and MS. All of these proteins will be analyzed using Panther and IPA to identify the relationship in host cell’s different metabolic pathways. 5-6 proteins involved in cell survival, antiviral or viral replication will be selected to analyze their effect in IAV growth after knocking down or over expression in cell lines. Conclusion: Three of our five newly developed mAb’s epitopes cover 97% of human IAV NS1 sequences among 1800 non-redundant IAV NS1 protein sequences of the NCBI Influenza Virus Resource database. I strongly believe that highest number of NS1 interacting host factors will be detected, where certain subsets of proteins may be discovered that affect IAV replication. Vasculogenic Mimicry in Placenta: Understanding Formation of Maternal Blood Vessels by Trophoblast Giant Cells Anshita Rai1, M. Gasperowicz2, and J.C. Cross1,2

1Department of Placenta is an organ that connects foetus to mother during pregnancy. It is crucial for nutrient and oxygen Biochemistry and exchange. It has a unique circulatory system, proper functioning of which is significant for successful pregnancy outcomes. Defects in placenta leads to various pregnancy related complications such as fetal Molecular Biology, death, abruption placentae, miscarriage etc. Studies have shown that placental malfunctioning might Faculty of Medicine, also have long-term health issues in child an mother. Due to genetic, physiological and morphological 2Department of commonalities with human, mice is an efficient model to study placentation. In mouse placenta, trophoblast giant cells (TGC) generate vessel-like structures without participation of endothelial cells that line blood Comparative Biology vessels throughout placenta. This phenomenon of vasculogenic mimicry is interesting but not well and Experimental understood. Previously, our lab has determined that TGCs have different phenotypes at various levels of the Medicine, Faculty of vascular circuit. The aim of my project is to develop a 3-dimensional in vitro murine trophoblast cell culture model and use this model along with in vivo system to unravel the phenomenon of vasculogenic mimicry. Veterinary Medicine, In order to develop a 3D model, I cultured trophoblast stem cells in suspension to develop spheroids (3D University of Calgary, trophospheres). These trophospheres had cavities similar to the maternal blood spaces formed by TGCs in Calgary, AB vivo. Histological, morphological and gene expression analysis confirmed that the cavities in trophospheres are imitation of vascular spaces in the venous side of placenta. Further, to unravel the mechanism behind vasculogenic mimicry, I exposed trophosphere culture to VEGF and hypoxia. Interestingly, I observed a dramatic change in expression of genes associated with TGCs. Also analysis of placenta and trophospheres by electron microscopy pointed some novel junctional interactions between TGCs and endothelial cells. However, further analysis is required to understand the detailed role of VEGF and adhesion molecules in establishing pseudo- vascular spaces. Overall our data suggest that the 3D trophospheres culture is a good model to study effects of both genetic and non-genetic factors on vascular development of placenta in relatively short time. It might also offer a wide range of opportunities to study both physiological and pathological events in placenta. Hence developing a model for studying vasculogenic mimicry will help us answer many questions of medical significance. Resveratrol Alleviates Oxidative Stress in Adult Cardiomyocyte by Preserving the Antioxidant Enzyme Activity P. Raj, A. Movahed, L. Yu, S.J. Thandapilly, X.L. Louis, S. Zieroth, T. Netticadan

Canadian Centre for Oxidative stress is a leading factor contributing to the initiation and progression of heart disease. The Agri-Food Research in imbalance in the oxidative status of the cell is mainly due to the malfunctioning of cellular antioxidant defense system. Resveratrol (trans-3,5,4’-trihydroxystilbene) is a plant polyphenol present grapes, peanuts, Health and Medicine, and berries, which has been shown to protect against a wide range of diseases. Our earlier studies have Winnipeg, MB, showed that resveratrol reduced oxidative stress and protected the cardiac function in different in-vivo Agriculture and heart disease models. The current study was performed to elaborate the role of resveratrol in mitigating oxidative damage and understand the underlying mechanism of its action by using a well established Agri-Food Canada, in-vitro model of oxidative stress. Adult rat cardiomyocytes were pre-treated with or without resveratrol, Winnipeg, MB, and then exposed to hydrogen peroxide (H2O2). Cell injury, cell death, oxidative stress and the activities Department of of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were assayed in control and H O exposed cardiomyocytes which were treated with and without resveratrol. Inhibitors of Physiology, University 2 2 antioxidant enzymes were used to confirm their role in mediating resveratrol action in 2H O2 exposed of Manitoba, cardiomyocytes. Cell injury and death, as well as decreased SOD and CAT activities were observed in H2O2 Winnipeg, MB exposed cardiomyocytes in comparison to controls whereas GPx activity was not affected. Pre-treatment of cardiomyocytes with resveratrol prevented the H2O2 induced oxidative stress as evidenced by the reduced cell death and injury as well as the increased activity of antioxidant enzymes. Pharmacological blockade of SOD and CAT negated the beneficial effect of resveratrol in 2H O2 exposed cardiomyocytes. To conclude, resveratrol prevents oxidative stress induced injury and death of cardiomyocytes by preserving the activities of key antioxidant enzymes and this may contribute to resveratrol mediated cardioprotection. Zinc and Diabetes: Examining the Effects of Zinc in Zucker Diabetic Fatty Rats Leslie Rech and Carla Taylor

Department of Human Diabetes is the most common endocrine disease worldwide, with type 2 diabetes accounting for 90% of Nutritional Sciences, cases. Low zinc status is common in people with diabetes and contributes to hyperglycemia, leading to hypozincemia and hyperzincuria. Zinc is involved in diabetes since it is required for insulin production, University of Manitoba, storage and secretion, and regulates the metabolism of adipose tissue. Many of the symptoms of zinc and Canadian Centre deficiency and diabetes are the same, and are related to increased inflammation; chronic inflammation has for Agri-Food Research been associated with reduced circulating zinc concentrations. Therefore, altered zinc homeostasis, which is present in diabetes, may contribute to inflammation and may exacerbate diabetes characteristics. The in Health and purpose of this study was to determine if marginal zinc deficiency (MZD) worsens and zinc supplementation Medicine, St. Boniface (ZS) improves parameters of diabetes and inflammation in Zucker diabetic fatty (ZDF) rats. The study used Research Centre, 30 male ZDF rats fed MZD (3 ppm), zinc control ([ZC], 30 ppm), or ZS (300 ppm), and 10 male lean ZDF rats fed ZC diet, for 8 weeks. Assessment of diabetes included obesity, hyperlipidemia, hepatic steatosis, oral Winnipeg, MB glucose tolerance testing (OGTT), glycemic control, and pancreatic islet insulin immunostaining. Systemic inflammation was assessed using serum haptoglobin and monocyte chemoattractant protein-1 (MCP- 1). Results showed ZDF rats fed ZC exhibited hyperlipidemia, hepatic steatosis, hyperinsulinemia, insulin resistance, and a higher area-under-the-curve during OGTT, although, they did not develop hyperglycemia, compared to lean ZDF rats. ZDF rats fed MZD had significantly reduced peri-renal fat pad mass and improved blood lipid levels (increased high-density lipoprotein cholesterol and decreased triglycerides); however, they had reduced glucose tolerance and β-cell function due to pancreatic atrophy and β-cell hypertrophy compared to ZC. ZS contributed to pancreatic hypertrophy without changing the number or size of β-cells. Obesity and diabetes elevated serum concentrations of haptoglobin and MCP-1, although, there was no effect of zinc deficiency or supplementation on these markers of systemic inflammation. Overall, MZD may exacerbate diabetes, while ZS had no effect on diabetes in ZDF rats. Evolution of Polyfunctional and Poliferative CD8+ T Cell Responses from Early to Chronic HIV-1 Infection1 Meika Richmond1, Sandra A. Koesters Kiazyk2, Lyle R. McKinnon3,4, Charles Wachihi4, Makobu Kimani4, Joshua Kimani1,4, Francis A. Plummer1,4,3 and T. Blake Ball1,2,4,5,6

1Department of The limited success of HIV vaccine candidates to date highlights our need to better characterize protective Medical Microbiology, cell-mediated immunity (CMI). Subgroups of HIV-infected subjects, long-term non-progressors (LTNP) and Elite Controllers (EC), experience slower progression to AIDS, by maintaining high CD4+ T cells and/or low University of Manitoba, viral loads, and provide a valuable model for the study of CMI responses that may be capable of controlling 2 National Laboratory HIV. Previous work has demonstrated that these individuals maintain stronger HIV-specific CD8+ T cell for HIV Immunology, proliferation compared to progressing controls. LTNP and EC were also found to have more polyfunctional Public Health Agency HIV-specific CD8+ T cells, as defined by the concurrent expression of cytokines IFN-γ, IL-2, and TNF-α, the of Canada, Winnipeg, chemokine MIP-1β, and the degranulation marker CD107a. However it is unclear whether these CD8+ T cell characteristics are responsible of the better disease outcomes or if they are merely a consequence of the 4 MB, Department of individuals high CD4+ T cells and low viral loads as a result of protection other means. Here we assessed the Medical Microbiology, evolution of CD8+ polyfunctional and proliferative responses from early to chronic HIV-1 infection in 26 HIV University of Nairobi, infected individuals following seroconversion. We hypostasizes that CD8+ T cells will become increasingly Kenya, 3Department of polyfunctional and proliferative from early to chronic infection and patients that mount and maintain an early polyfunctional and proliferative response will have a better disease outcome. Our data suggests Medicine, University of that the polyfunctional and proliferative capacity of CD8+ T cells from early to chronic infection follows a Toronto, Toronto, ON, distinct pattern, and further that CD8+ T cell responses vary substantially between individuals in the chronic 5National Microbiology phase of infection. Case study analysis of individuals CD8+ T responses over time suggests that individuals Laboratory, PHAC with moderate polyfunctionality in the early phase of infection go one to maintain healthy CD4+ T cell counts. Identification of whether polyfunctional responses and strong proliferative capacity is the cause or 6Department of consequence of HIV control will be needed for the comprehensive evaluation of HIV vaccine candidates. Immunology, University of Manitoba, Winnipeg, MB Characterizing Nutrition Status in a Manitoba First Nation Community Natalie D. Riediger, Sharon Bruce

Department of Background: Diabetes and its associated co-morbidities are major contributors to population morbidity Community Health and premature mortality. While the descriptive epidemiology of diabetes has been well described, the pathways between risk factors and conditions and disease outcomes are less well understood. Dietary Sciences, Faculty of transition occurred rapidly among First Nation people and has contributed to increasing rates of diabetes Medicine, University of and other chronic diseases. However, the transition has not been well characterized at the population level. Manitoba, Winnipeg, Such characterization is necessary in order to identify relevant targets for intervention. The purpose of this study is to characterize nutrition status and factors affecting food choices among members of one Manitoba MB First Nations community. This study is embedded within a larger population-based study on diabetes epidemiology. Methods: A population-based concurrent mixed methods study using a community-based participatory research framework. All adult members of Sandy Bay First Nation were invited to participate. Data collection occurred from July 2011-August 2012, and included a 24-hour dietary recall and Dietary Questionnaire. Dietary recall data are linked to the Canadian Nutrient File (2010) and additional data were constructed using Canada’s Food Guide, the USDA Added Sugar Database, and traditional aboriginal knowledge. These data are linked to participant data from the larger Stress & Diabetes Study, including blood pressure, anthropometrics, biological data from blood samples, and administrative health records. Qualitative interviews were also conducted among 21 community members. Lastly, Food Environment data were collected from three grocery stores and two convenience stores, including food availability, cost, and quality. Results: 473 participants completed the 24-hour dietary recall and First Nation Diet Questionnaire. Of those participants, 344 also completed the protocol for the Stress & Diabetes Study. All age and sex groups were well-represented. Traditional food intake remains common in the community, particularly traditional meats. Food insecurity is common and is associated with self-rated diet (i.e., higher food insecurity was associated with poorer perceptions of diet). Conclusions: Data entry, analysis, and knowledge translation activities with the community are ongoing. This rich dataset will allow a comprehensive understanding of the nutrition environment in Sandy Bay First Nation and subsequently inform a diabetes intervention in the community. Systems Biology Analysis of Human Mucosal Fluids Shows a Unique Environment for HIV-Infection in Rectal Mucosa Laura M. Romas1, Kenzie D. Mogk1,4,Lindsay G. Aboud, Klara Hasselrto2, Richard Novak5, Stuart McCorrister4, Garrett R. Westmacott4, Francis A. Plummer1,4, Blake T. Ball1,4, Kristina Broliden2, Adam D. Burgener4

1University of Manitoba, Introduction: Sexual exposure of HIV occurs at the oral, cervicovaginal and rectal mucosae, but HIV- Winnipeg, MB, transmission is heterogeneous between sites. Protein factors associated with HIV pathogenesis have been identified in cervicovaginal and oral mucosae; however, knowledge of immune factor expression 2 Karolinska Institute, in the rectal mucosa, and distribution patterns between compartments is lacking. This represents a Sweden, 3University of major limitation in our understanding of HIV-susceptibility associated with the high-risk practice of Nairobi, Kenya, 4Public receptive anal intercourse, as well as our ability to design compartment-specificprevention strategies, such as anti-HIV microbicides. This study uses systems biology to characterize mucosal immune factor Health Agency of expression between sites of HIV exposure. Canada, Winnipeg, MB, Methods: Salivary fluid (SF, n=10), rectal lavage (RL, n=10) and cervicovaginal lavage (CVL, n=10) 5University of Illinois at fluid was collected from healthy individuals and analyzed by label-free tandem mass spectrometry. Proteomic data was further analyzed using a combination of differential protein expression analysis Chicago, Chicago, IL (one-way ANOVA, p<0.05; Perseus 1.3.0.4), hierarchical clustering (Cluster 3.0, JavaTreeview 1.16r2), and functional annotation (www.uniprot.org). The inhibitory capacity of mucosal fluidsagainst R-tropic HIV-Bal was assessed using a TZM-bl reporter cell line (one-tailed t-test, p<0.05). Results: Of the 314 proteins identified, 176 were differentially abundant between mucosal fluids (p<0.05). Hierarchical clustering showed similarity in protein expression in SF and RL relative to CVL. Further functional analysis of host immune factors, including antimicrobial proteins, antiproteases, and pro-/anti-inflammatoryproteins were underabundant in RL and SF compared to CVL (underabundant in >75% of all identified proteins), while immunoglobulins were uniquely enriched in RL (75% of all identified). Mucosal inhibition assays demonstrated salivary fluid(p<0.005) and, for the first time, rectal lavage fluid (p=0.05) to significantlyinhibit infection of TZM-blreporter cells by lab strains of HIV-Bal in vitro. Conclusion: Mucosal fluids vary significantly with respect to immune factor expression and HIV- inhibitory properties. This is likely a contributing factor to HIV transmission variability at these three mucosal sites of exposure and an important consideration in the design of HIV-prevention strategies/ therapeutics. The Use and Interpretation of Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) for Escherichia coli O157:H7 Outbreaks in Canada J. Rumore1,2, L. Tschetter2, K. Tabing2, C. Peterson2, C. Goodman2, A. Kearny2, C. Nadon1,2

1Department of Introduction: Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) is a molecular subtyping Medical Microbiology method for use with Pulsed-field Gel Electrophoresis (PFGE), the current gold standard method for subtyping Escherichia coli O157:H7. Ideally, MLVA should provide further specificity to outbreak and Infectious investigations by helping to differentiate outbreak and sporadic cases. The objective of this study was to Diseases, University of determine the usefulness of MLVA for the identification and investigation of three recent and separateE. coli Manitoba, Winnipeg, O157:H7 outbreaks in Canada. Methods: Using the subtyping methods PFGE and MLVA, a descriptive analysis of three recent and separate MB, 2National E. coli O157:H7 outbreaks associated with walnuts and beef products was conducted. Microbiology Results: 46 isolates were subtyped during the time period of a 2011 outbreak associated with walnuts; 14 Laboratory, Public were found with the PFGE pattern combination ECXAI.1194, ECBNI.0895, which was not common at that time. The MLVA results were useful in differentiating this outbreak from a concurrent outbreak associated Health Agency of with shelled hazelnuts in the United States. Of the 148 isolates analysed for a 2012 outbreak associated Canada, Winnipeg, with beef products, 18 outbreak-related cases were found to have the highly common PFGE pattern MB ECXAI.0001, ECBNI.0012. The MLVA results were integral in separating outbreak-related and sporadic cases, and for confirming the source of contamination. During a second 2012 outbreak associated with frozen beef burgers, 5 cases out of 139 isolates were confirmed with the PFGE pattern ECXAI.2850, ECBNI.0202, which has not been seen in Canada before. The MLVA results confirmed the findings of PFGE; however, PFGE was used to define the outbreak and to confirm the food source. Conclusion: The utility of MLVA was dependent on the prior frequency of the PFGE pattern of the outbreak strain; it provided specificity when the PFGE pattern was common. A thorough analysis of the distribution of MLVA profiles among surveillance and outbreak data in Canada is needed and is currently underway. Chemical Components of Common Stain Repellents Can Modulate Non-Allergic and Allergic Lung Inflammation and Hyperresponsiveness in Mice Min Hyung Ryu1,2, Aruni Jha1,2, Neda Nikoobaht3, Sujata Basu1,2, Mark Loewen2, Charles Wong3,4, Allan B. Becker2,5 Andrew Halayko1,2

1Department of Introduction: Pregnant mothers and children are exposed to many consumer products (e.g., stain Physiology, University repellents) containing perfluoronated compounds including perflurooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (FTOH), which is bio-converted to PFOA. PFOA modulates peripheral leukocyte of Manitoba, cytokine synthesis. This affects adaptive immunity that can affect susceptibility for diseases such as asthma. 2Manitoba Institute of We evaluated whether in utero-through-postnatal exposure of mice to PFOA or FTOH impacted basal and Child Health allergic lung inflammation and function. Methods: Pregnant Balb/c mice were exposed to ingested PFOA (4mg/kg chow) from gestation day (GD) 3Department of 2 until weaning. Pups ingested PFOA-chow until 12 weeks of age. A second cohort of mice was housed Chemistry, University with FTOH-coated huts from GD14, pups remaining in this environment during and after weaning. Some of Manitoba, pups were also sensitized and challenged with ovalbumin (OVA) in the final 3 weeks of chemical exposure. 4Richardson College Serum PFOA was analyzed by high performance LC-MS-MS. At 12 weeks of age we assessed lung function using flexiVent small animal ventilator. Bronchoalveolar lavage fluid (BALF) was collected to assay total for the Environment, inflammatory cell number and distribution. Lungs were paraffin fixed and assessed for morphological University of Winnipeg, changes using hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) stain. Winnipeg, MB, Results: In PFOA- and FTOH-exposed mice serum PFOA reached 4800 1100 ng/mL and 5400 900 ng/mL, respectively. Only PFOA-exposed mice exhibited lung inflammation, chiefly consist of macrophages. Total 5 Department of BALF cell count was 25-fold higher than for FTOH-exposed and naïve mice (P<0.05). Nonetheless, both Pediatrics and Child PFOA- and FTOH-exposed mice exhibited airway hyperresponsiveness (AHR) to methacholine (64.1% and Health, University of 66.3% lower PC100 than naïve mice; 53% and 70% greater airway resistance at 50mg/ml methacholine exposure than naïve mice; P<0.05). PFOA ingestion had no impact on OVA-induced lung inflammation and Manitoba, Winnipeg, AHR, but FTOH blunted OVA-induced inflammation. MB Conclusion: In utero and postnatal exposure to ingested PFOA increases lung macrophages and AHR, but does not predispose for enhanced allergic lung inflammation or AHR. PFOA exposure from bioconversion of inhaled FTOH suppresses allergic lung inflammation and AHR. Our data implicates complex biological mechanisms linking environmental toxin exposure with susceptibility to acquire an asthma phenotype. Hypoxia Activated Pro-Drugs: A Novel Approach for Cancer Treatment Jasdeep K. Saggar and Ian F. Tannock

Departments of Introduction: Hypoxia is associated with treatment failure. Tumor cells closest to blood vessels are Medicine and Medical well nourished and rapidly proliferating but hypoxic cells located farther away are slowly-proliferating. Chemotherapy targets rapidly proliferating cells and therefore may spare hypoxic cells because of poor Biophysics, Princess drug distribution to them. Intervals between administration of chemotherapy allow normal tissues to Margaret Hospital and recover but might allow re-oxygenation and resumed proliferation of formerly hypoxic cells due to better University of Toronto, supply of nutrients to them. Hypoxia activated pro-drugs (i.e. TH-302) may inhibit tumor repopulation by killing hypoxic cells. Here we evaluate these processes in human tumors xenografts. Toronto, ON Methods: MCF7 tumor bearing mice were administered two specific markers of hypoxia (pimonidazole [pimo] and EF5). Hypoxia-labeled tumor cells were recognized in tumor sections (in relation to DioC7+ve functional tumor blood vessels) using immunohistochemistry. Proliferating cells were identified by an antibody to Ki67. Mice were treated with pimo and: saline, doxorubicin (DOX), TH-302 or TH-302 + DOX; EF5 was given after a variable interval of 24, 48, 72, 96 or 120 hours and tumors collected. Changes in the proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their Ki67 status and uptake of EF5 as a function of time. Results: Following DOX treatment, the proportion of hypoxic cells in the entire tumor decreased from 1.5% (pimo+ve) prior to injection to 0.7% (EF5+ve) at 24 hours. 90% of pimo+ve formerly hypoxic cells were no longer hypoxic (i.e. EF5 – ve) at 24 hours after doxorubicin compared to 27% in controls, indicating rescue of previously hypoxic cells that would have died in the absence of treatment. Proliferation of these pimo+ve cells that were cycling (Ki67+ve) increased from 7% to 15.0% at 24 hours and then slowly decreased. TH-302 + doxorubicin combination inhibited repopulation by reducing Ki-67 to 1%. Conclusions: Formerly hypoxic cells in MCF7 xenografts undergo re-oxygenation and increase their proliferation following treatment with doxorubicin. Originally hypoxic cells (that may have died in the absence of treatment) can move closer to blood vessels , re-oxygenate & repopulate a tumor. TH-302 can inhibit hypoxic cell proliferation & tumor repopulation, thereby increasing chemotherapeutic outcome. Synthetic Lethal Killing of RAD54B-deficient Colorectal Cancer Cells by Targeting Superoxide Dismutase 1 Babu V. Sajesh, Zelda Lichtensztejn, and Kirk J. McManus

Manitoba Institute of Introduction: Colorectal cancer has a significant health burden and thus novel therapeutic strategies are in Cell Biology & dire need. Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations in genes that cause chromosome instability. RAD54B is required Department of to maintain chromosome stability and is an excellent candidate for therapeutic intervention as somatic Biochemistry and mutations have been identified in numerous tumor types. Consequently, therapeutic strategies designed to Medical Genetics, exploit RAD54B-deficiencies will likely have broad-spectrum applicability. Methods: Using cross-species approaches, we identified a candidate synthetic lethal interaction between University of Manitoba, human RAD54B and SOD1, an enzyme that functions in the removal of DNA-damaging reactive oxygen Winnipeg, MB species. We further employed high-content imaging, RNAi mediated silencing and chemical inhibition to confirm and validate theRAD54B SOD1synthetic lethal interaction. Results: We show that RAD54B-deficient cells are selectively killed relative to controls following siRNA- based silencing of SOD1. Two chemicals ATTM, a SOD1 inhibitor, and 2ME2, an inducer of reactive oxygen species, could functionally substitute for the SOD1 siRNAs and recapitulate this synthetic lethal interaction. Standard dose-response curves indicate that RAD54B-deficient cells were hypersensitive to ATTM and 2ME2 treatments, and was further validated by colony formation assays. Finally, high-content imaging microscopy was employed to evaluate the mechanism of death. Statistically significant increases in the global abundance of DNA double-strand breaks as identified by semi-quantitative increases inγ -H2AX and 53BP1 signal intensities were observed within the RAD54B-deficient cells treated with ATTM and 2ME2, which correlated with significant increases in apoptosis as measured by increases in cleaved Caspase 3 intensities. These data demonstrate that the RAD54B SOD1 synthetic lethal interaction is evolutionarily conserved and thus validate SOD1 as a novel candidate therapeutic target. Conclusions/Future Directions: Our findings rationalize using cross-species approaches to identify novel therapeutic targets, for selective and broad-spectrum targeting of cancer. Identification of SOD1 as a candidate target is significant consideringRAD54B is somatically mutated in several tumors (colorectal, breast, glioblastoma, etc.). Epidemiological and Descriptive Characteristics of Oral Health Systematic Reviews Published 1991-2012 Humam Saltaji1, Greta G. Cummings2, Maryam Amin1, and Carlos Flores-Mir1

1Department of Objectives: To identify all systematic reviews (SRs) published in the domain of oral health research and Dentistry, University of describe them in terms of epidemiological and descriptive characteristics, and to develop a registry of all oral health SRs. 2 Alberta, Faculty of Methods: An electronic search of seven databases was performed from inception through May 2012. Nursing, University of Studies were considered for inclusion if they were oral health SRs defined as therapeutic or non- Alberta, Edmonton, AB therapeutic investigations that studied a topic related to dental, oral or craniofacial diseases/disorders. Data were extracted from all the SRs on a number of key epidemiological and descriptive characteristics. Data were analysed descriptively for all the SRs, within each of the dental specialities, and for Cochrane and non-Cochrane SRs separately. Results: 1188 oral health (126 Cochrane and 1062 non-Cochrane) SRs published from 1991 through May 2012 were identified, encompassing nine dental specialties. Over half (n = 676; 56.9%) ofthe SRs were published in specialty journals, with almost all (n = 1178; 99.2%) of the SRs published in English and almost no non-Cochrane SRs (n = 11; 0.9%) were updates of previously published SRs. Corresponding authors were more frequently from Europe followed by North America, with four countries (USA, UK, Canada, and The Netherlands) accounting for nearly half (48.9%) of all oral health SRs. 75.3% of the SRs were categorized as therapeutic, with 64.5% examining non-drug interventions. The SRs included a median of 15 studies, with a meta-analysis conducted in 43.6%, in which a median of 9 studies/1 RCT were included in the largest meta-analysis conducted. Conclusion: Epidemiological and descriptive characteristics of the 1188 oral health SRs varied across the nine dental specialties and by the SR category. There is a clear need for more updates of SRs in all the dental specialties, and for more SRs in the field of oral and maxillofacial radiology. Enforcement of a Lifespan-Sustaining Distribution of Sir2 between Telomeres, Mating- Type Loci, and rDNA Repeats by Rif1 Jayesh S. Salvi1, Janet N.Y. Chan1, Christopher Pettigrew1, Tony T. Liu1, Jane D. Wu1, and Karim Mekhail1

1Department of Introduction: Dysfunction of telomeres or their interacting factors is linked to genome instability and Laboratory Medicine premature aging. Functions of sirtuin proteins, which are linked to various aging processes, at telomeres are thought to promote lifespan in yeast and mammals. However, replicative lifespan of the budding yeast and Pathobiology, Saccharomyces cerevisiae shortens upon deletion of Rif1, a protein that limits the recruitment of the sirtuin University of Toronto, histone deacetylase Sir2 to telomeres. Here, we show that Rif1 maintains replicative lifespan by ultimately Toronto, ON stabilizing another age-related chromosomal domain harboring the ribosomal DNA (rDNA) repeats. Methods/Results: Deletion of Rif1 increases Sir2 localization to telomeres and the silent mating-type loci while releasing a pool of the histone deacetylase from the intergenic spacer 1 (IGS1) region of rDNA repeats. This is accompanied by a disruption of silent chromatin assembly at IGS1 and an increase in aberrant recombination events within rDNA repeats. Lifespan defects linked to Rif1 deletion can be prevented in settings where rDNA repeats are forcibly stabilized via deletion of the replication fork blocking protein Fob1. Moreover, Sir2 over-expression prevents Rif1 deletion from excessively reducing Sir2 localization/function at IGS1 and shortening replicative lifespan. In addition, subjecting cells lacking Rif1 to caloric restriction reduces IGS1 histone deacetylation, increases replicative lifespan, and reveals genetic interactions between Rif1 and Sir2 in the cellular response to changes in nutrient levels. Conclusions: Our data indicate that Rif1 maintains lifespan-sustaining levels of Sir2 at rDNA by preventing the excessive recruitment of the histone deacetylase to telomeres and silent mating-type loci. Since sirtuin histone deacetylases such as Sir2 or mammalian SIRT6 each operate at multiple age-related loci, we propose that factors limiting the localization of sirtuins to certain age-related loci can promote lifespan-sustaining roles of these sirtuins elsewhere in the genome. RTEL1 DNA Helicase is Required for the Maintenance of Spermatogonial Stem Cells Sumit Sandhu, Xiaoli Wu, Wenjun Liu, Hao Ding

Department of RTEL1 (Regulator of Telomere Length 1) is a DNA helicase which has been demonstrated to be essential Biochemistry and for the maintenance of telomere length and genomic stability. However, whether this function of RTEL1 is important for development is still largely unknown. In this study, we found that RTEL1 is specifically Medical Genetics, expressed in spermatogonial stem cells (SSCs), a group of stem cells required for spermatogenesis. Using University of Manitoba, the combined lineage-tracing and stem cell-transplantation assays, we further demonstrated that RTEL1+ Winnipeg, MB SSCs can self-renew and differentiate to spermatocytes. In order to determine the role of RTEL1 in SSCs, we applied a conditional knockout approach to inactivate RTEL1 function in these cells. We observed a significant decrease in the testis size in the knockout group as compared to control. Histological examinations revealed compromised self-renewal and differentiation in RTEL1 knockout SSCs. Moreover, on p53 null background, RTEL1-deficient SSCs were found to grow as testicular tumors. Taken together, my study indicates that RTEL1 is required for the maintenance of SSCs most likely by protecting their genomic integrity, and this function could be important for preventing these stem cells being transformed. Multi-state Markov Model for Prostate Cancer Management Chirandjeev Sanyal1,2, Armen G. Aprikian1,2, Fabio L. Cury3, Simone Chevalier1,2, Alice Dragomir1,2

1Research Institute of Introduction: Prostate cancer (PCa) is the most common cancer among Canadian men with an incidence McGill University of 121 cases per 100,000 individuals in 2011. The incidence of PCa has increased significantl y over the years; however, mortality rates are relatively steady due to increase in the detection of low-risk PCa and Health Center, effective management of the disease. Therefore, the prevalence of PCa is expected to increase leading to 2Department of substantial burden on the healthcare system. The aging population magnifies the burden of PCa on the Surgery, Division of healthcare system since most of the cases are diagnosed in men 60 years and older. In 1997, the lifetime direct healthcare cost of PCa management in Canada was estimated at $9.8 billion. In addition to existing Urology, McGill PCa interventions (i.e. radical prostatectomy, external beam radiotherapy, and androgen deprivation University, therapy), the publicly funded healthcare system in Quebec, Canada has adopted newer health technologies 3Department of such as robotic-assisted radical prostatectomy and intensity-modulated radiation therapy to improve health outcomes and to minimize post intervention complications. Oncology, Division of Objectives: The proposed study would develop and validate a multi-state Markov model with first-order Radiation Oncology, Monte-Carlo simulations representing the natural course of PCa management. McGill University, Methods: A multi-state Markov model with Monte-Carlo microsimulations would simulate the natural Montreal, QC course of PCa management. PCa would be represented by six discrete states: (I) active surveillance, (2) initial treatment, (3) prostate specific antigen (PSA) relapse, (4) PSA relapse free, (5) metastatic PCa, and (6) death. The model would consider following management options: active surveillance, open/robotic radical prostatectomy, intensity modulated radiotherapy (IMRT), brachytherapy, androgen deprivation therapy (ADT), IMRT combined with ADT, and chemotherapy. The state transition probabilities would be abstracted from peer reviewed articles. The validation of the model would be conducted by comparing state transition probabilities predicted by the model to the published literature. Conclusion: The proposed model could predict clinical outcomes and healthcare expenditure by PCa treatment options. Therefore, this model could be used for healthcare decision making. Investigating the Role of Protein S as a Pro-Survival Factor during Prostate Cancer Progression Punit Saraon1,2, Keith A. Jarvi4, Eleftherios P. Diamandis1,2,3

1Laboratory Medicine Introduction: Androgen-deprivation remains the principal therapy for advanced and metastatic prostate and Pathobiology, cancers. However, some cancer cells can survive this treatment and transform themselves to a more aggressive androgen-independent prostate cancer (AIPC). An understanding of the molecular alterations University of Toronto, that occur during the progression to androgen-independence is an integral step to generate effective 2Samuel Lunenfeld targeted therapies. Research Institute, Methods: Using Mass Spectrometry, we identified Protein S (PROS1) to be specifically elevated in androgen-independent cell lines (PC3, DU145, PPC1, LNCaP-SF, 22Rv1) compared to androgen-dependent Mount Sinai Hospital, (LNCaP, VCaP) and normal prostate epithelial (RWPE) cell lines. Tissue validation using qPCR and 3Department of immunohistochemistry analysis revealed elevation of PROS1 during high grade prostate cancer (Gleason Clinical Biochemistry, ≥ 8), and further elevation in castrate-resistant metastatic prostate cancer lesions. We also observed its elevation in high grade prostate cancer seminal plasma samples (P<0.05). Mount Sinai Hospital, Results: To understand the functional role of PROS1 with respect to prostate cancer progression, we Toronto, ON, generated stable PROS1 knock-downs in DU145 cells and over expression PROS1 cells in LNCaP cells, 4Department of and performed cell migration and viability assays. Cell migration and proliferation assays revealed that Surgery (Division of PROS1 enhanced growth of prostate cancer cells, as there was significantly reduced wound closure in shPROS1 DU145 cells compared to scrambled control cells (p<0.05). In addition, cell viability assays Urology), University of showed that shPROS1 cells had reduced survival rates compared to scrambled cells when treated with the Toronto, Toronto, ON chemotherapeutic agents, docetaxel and paclitaxel, indicating its role as a potential anti-apoptotic factor. We also observed that the PI3K/AKT pathway was responsible for inducing an anti-apoptotic cascade through the elevation of the anti-apoptotic factors Bcl-2 and Bcl-xl. Conclusions: Taken together, our results show that PROS1 is elevated during high grade and castrate- resistant prostate cancer, and promotes prostate cancer migration and cell survival. The Tumor Necrosis Factor Alpha/Sphingosine-1-Phosphate Signalling Axis Mediates Augmentation of Microvascular Tone in a Mouse Model of Type 2 Diabetes Mellitus M. Sauvé and S.S. Bolz

Department of Diabetes is a primary risk factor for cardiovascular disease, and is associated with increased mortality due to Physiology, Faculty of the complications of widespread end-organ damage (e.g. renal dysfunction, heart failure, cerebrovascular ischemia). The pre-capillary resistance arteries, key regulators of tissue perfusion, are targets of metabolic Medicine, University of dysregulation. Compromised endothelial signaling has been implicated as a cause of microvascular Toronto, Toronto, ON dysfunction, however, this approach has not yet yielded effective therapies. This may result from the role for microvascular smooth muscle cells (MVSMCs) as direct targets of diabetes-induced pathological processes. MVSMCs convert transmural pressure into biochemical signals to regulate contractility, termed the myogenic response. Sphingosine-1-phosphate (S1P) is a central regulator of the myogenic response through the pressure-induced activation of the S1P producing enzyme sphingosine kinase 1 (SphK1) and the S1P2 receptor. Recently, TNFa has been shown to augment the myogenic response of resistance arteries in diseases that share an inflammatory component (e.g. heart failure). As diabetes is associated with increased TNFa, we propose that activation of the TNFa/S1P signaling axis contributes to microvascular dysfunction in diabetes. Mice were treated with high fat diet (HFD) + streptozotocin to induce type 2 diabetes (T2D). T2D mice had elevated HbA1C levels compared to HFD controls. T2D mice show augmented myogenic tone in isolated mesenteric, posterior cerebral, and olfactory resistance arteries, while resistance arteries isolated from HFD mice did not display altered myogenic tone even after one year on HFD. T2D did not alter artery responses to phenylephrine. These data suggest that hyperglycemia/diabetes, not obesity, specifically alters the myogenic response in MVSMC. TNFa was implicated, as acute in vitro treatment with the TNFa scavenger etanercept attenuates the augmented myogenic response in isolated resistance from T2D mice. Chronic TNFa inhibition by 1) in vivo etanercept treatment or 2) genetic deletion of TNFa also normalized the myogenic response in T2D. A critical role for S1P was implicated as elevated myogenic tone in T2D was normalized by the S1P2 receptor antagonist JTE013 and in resistance arteries from diabetic SphK1-/- mice. Our data suggest that the TNFa/S1P signaling axis regulates diabetes-induced augmentation of the myogenic response and identifies potential therapeutic targets to mitigate microvascular dysfunction in diabetes. Estrogen Regulation of Anti-Apoptotic Bcl-2 Family Member, Mcl-1 Expression in Breast Cancer Jennifer L. Schacter1,2 , Elizabeth Henson2, and Spencer B. Gibson1,2

1University of Objectives: Estrogen is implicated as an important factor in stimulating breast cancer proliferation, and Manitoba, Winnipeg, presence of estrogen receptor (ER) is an indication of good prognosis. Mcl-1 is an anti-apoptotic Bcl-2 family member that is often over-expressed in breast tumours, correlating with poor survival. I have investigated 2 MB, Manitoba the role estrogen plays in regulating Mcl-1 expression. Ultimately, I will determine if estrogen regulates Mcl- Institute of Cell Biology, 1 expression through a mechanism involving its receptor ERα. Winnipeg, MB Methods: Two distinct breast cancer cell lines were used: MCF-7 and ZR75, which both express ERα. Cells were grown in serum-starved white media with charcoal-stripped FBS for five days prior to treatment with estrogen (10 nM) over a 24-hour time course. Afterwards, cells were lysed and western blotted for Mcl-1 protein expression. I determined the level of mRNA for the full-length transcript by quantitative real time PCR following estrogen treatment. Cells were also treated with ER antagonists Tamoxifen (200 nM) and ICI- 182,780 (500 nM) over a 24-hour time course. Following treatment, western blot and quantitative real time PCR were performed similar to previous experiment. In order to determine the role of ERβ, MCF-7 cells were treated with a Tet-On that overexpresses ERβ in the presence of doxycycline. As well, MDA MB 231 clone 88, a stable breast cancer cell line that overexpresses ERβ, and its parental clone MDA MB 231 clone 40, were treated with estrogen (10 nM) over a 24-hour time course. In order to determine the role of ERα, knockdown of ERα was performed by electroporating 10 nM pooled ERα siRNA into MCF-7 cells and mRNA expression was measured after estrogen treatment. Results: In both MCF-7 and ZR75, estrogen treatment increased Mcl-1 expression at both the mRNA and protein level. Estrogen Receptor antagonists Tamoxifen and ICI-182,780 decreased Mcl-1 expression. Overexpression of ERβ in MCF-7 and MDA MB 231 cells appears to repress Mcl-1 protein expression. Knockdown of ERα decreases Mcl-1 mRNA expression after estrogen treatment. Conclusion: Estrogen could be involved in regulating Mcl-1 expression through ERα, yet the mechanism remains unknown. In the future, I will determine the role of ERα in Mcl-1 expression. Molecular Evolution of Measles Virus during the 2011 Outbreak in Quebec H. Schulz 1,2, J. Hiebert 1, J. Beirnes 1, L. Mendoza 1, G.A. Tipples 1,3, A. Severini 1,2

1National Microbiology Background: Endemic transmission of the highly infectious Measles virus (MeV) has been declared Laboratory, Public interrupted in Americas in 2002 and all cases are due to importations from endemic areas. Molecular surveillance with World Health Organization (WHO) standardized targets is important to document the non- Health Agency of endemic status of measles in the Americas. However, in 2011 several importations of identical D4 strains Canada, Winnipeg, MB, from Europe challenged the ability of molecular tests to distinguish imported from locally transmitted cases. 2Department of Taking advantage of a large Quebec outbreak, we determined the mutation rate of MeV during transmission over 10 month. Medical Microbiology, Methods: Clinical specimens were genotyped by RT-PCR amplification and sequencing of 450 nucleotides University of Manitoba, of the nucleoprotein (N) and the entire hemagglutinin (H) gene open reading frame. For whole genome Winnipeg, MB, sequencing, the isolates were propagated in cultures of Vero/hSLAM cells, the purified RNA was reverse transcribed and amplified by PCR in seven overlapping fragments. 3Provincial Laboratory Results: The 119 specimens from the Quebec outbreak were all identical in the 450nt WHO target sequence for Public Health, on the N-gene. Ten D4 strains detected from different importations in Canada also had identical sequence Alberta Health in the N-gene target region. In order to differentiate these strains, we sequenced the 1,854 nucleotides of Services, Edmonton, AB the H-gene, a secondary target recommended by WHO. All H-gene sequences from the Quebec outbreak were identical, while differentiating mutations were observed in the strains from outside Quebec. It is clear that the MeV mutated very slowly during the Quebec outbreak and therefore we sequenced the entire genome of these isolates in order to measure the mutation rate during the outbreak. Eleven entire genomes have been sequenced to date. Their alignment shows a very high degree of identity. Four genomes were completely identical and the average nucleotide difference per site was 3.99x10^-4 or nucleotide difference per 15,894nt genome was about 6. Conclusion: Our results suggest that the MeV genome changes very slowly during outbreaks, unlike the case for other RNA viruses. The WHO standardized regions on the N- and H-genes are not diverse enough to discriminate between outbreaks and new targets must be found in order to distinguish between direct transmissions and separate MeV introduction events. Systematic Analysis of HIV-1 Nef Epitopes of Two HLA Class I Alleles Associated with Different Outcomes of HIV-1 Infection Elnaz Shadabi, T. Bielawyny, J. Tuff, R. Capina, J. Kimani, C. Wachihi, M. Kimani, T.B. Ball, F.A. Plummer, Ma Luo

Medical Microbiology, In the Pumwani Sexworker Cohort, established in 1985 in Nairobi Kenya, a subset of women remain HIV- University of Manitoba, 1 uninfected despite repeated exposures through high risk sex work. In a previous study we showed that patients with HLA-B*07:02 are more susceptible to HIV-1 infection than those with HLA-A*01:01. In Winnipeg, MB, National this study we systematically analyzed HIV-1 clade A and D Nef CD8+ T cell epitopes of HLA-A*01:01 and Microbiology HLA-B*07:02 alleles, and their CD8+ T cell responses. The iTopia Discovery System was used to analyze Laboratory, Winnipeg, peptide binding, affinity and off-rate. IFN-gamma ELISpot assay with patient PBMCs was used to confirm epitopes capable of stimulating T cell responses. Our results showed that HLA-A*01:01 allele binds to three MB, Medical distinct HIV-1 Nef epitopes, whereas HLA-B*07:02 binds to 33 epitopes. The epitope binding affinity and off- Microbiology, rate are not significantly different between A*01:01 and B*07:02 (P = 0.2790 and P = 0.2557, respectively). University of Nairobi, However, unlike previous study of Gag epitopes, we did not detect IFN-gamma ELISpot responses with A*01:01 Nef epitopes in patient PBMCs. Among the 33 B*07:02 epitopes, we only detected IFN-gamma Kenya ELISpot responses to 19 peptides. The failure of detecting epitope specific IFN-gamma ELISpot responses in patient PBMCs could be due to low expression of Nef, since IFN-gamma ELISpot responses have been detected for more than 99% of the Gag epitopes identified by iTopia Discovery system in our previous study. Understanding T cell response and activation after HIV-1 infection is a crucial step towards implementing effective vaccine strategy. Reliability of a Uni-Compartmental Scale for the Radiographic Evaluation of Knee Osteoarthritis: Data from the Multicenter Osteoarthritis Study Lisa Sheehy1, T. Derek V. Cooke1, John Lynch2, Michael Nevitt2, Linda McLean1, Jingbo Niu3, Neil A. Segal4, Jasvinder Singh5, Elsie Culham1

1School of Introduction: Radiographs of the knee are used to determine the severity of knee osteoarthritis (OA) and Rehabilitation Therapy, monitor its progression. Commonly-used severity scales lack sensitivity to change or predominantly rely on one feature of knee OA. The unicompartmental osteoarthritis grade (UCOAG) is a composite scale that Queen’s University, grades four individual features of knee OA in the most-affected tibiofemoral compartment and sums them Kingston, ON, to create a total score. Because one feature is not emphasized all presentations of knee OA can be assessed 2Department of equally. The purpose of this study was to assess the intra-rater, inter-rater and test-retest reliability of the Epidemiology and UCOAG. Biostatistics, University Methods: Two samples of knee radiographs were selected from the Multicenter Osteoarthritis Study database (NIH-funded). Radiographs were stratified according to knee OA severity categories as seen on of California, San magnetic resonance imaging (MRI). Francisco, CA, 3Boston The first sample of 100 images was used to determine intra- and inter-rater reliability. Three readers graded University School of the 100 images twice using the UCOAG scale, two weeks apart. Medicine, Boston, MA, A second sample of 100 radiograph pairs, taken 15 or 30 months apart, was used for test-retest reliability. Knees without any increase in cartilage damage, as determined by MRI, were chosen. One reader graded 4Department of these images. Orthopaedics and Intraclass correlation coefficients (ICC2,1) and Cohen’s weighted kappa (κ) were performed for each type of Rehabilitation, reliability. Minimal detectable change (MDC90) was calculated using test-retest reliability data. University of Iowa, Results: From the first sample, ICCs for intra-rater reliability were 0.84 to 0.91 andκ were 0.65 to 0.75. For Iowa City, IA, inter-rater reliability the ICC was 0.79. κ between pairs of readers were 0.47 to 0.61. From the second sample, the ICC for test-retest reliability was 0.86. κ was 0.64. The MDC was 1.99. 5 90 University of Alabama Conclusion: The UCOAG has moderate to excellent intra-rater, inter-rater and test-retest reliability over at Birmingham School a large range of knee OA severity. These results are comparable or better than the reliability results of of Medicine, Birmingham, AB other scales used to grade knee OA on a radiograph. A change of two or more grades in the UCOAG scale indicates a true change in TF OA severity. The UCOAG is recommended to grade knee radiographs for knee OA severity, for clinical and research purposes. CD103+ Lung Dendritic Cells are Critical for Inducing Protective Immunity Against an Intracellular Bacterial Infection Sudhanshu Shekhar and Xi Yang

Laboratory for Introduction: Dendritic cells play a central role in the induction of protective immunity against infections. Infection and Lung dendritic cells (LDC) are comprised of two major subpopulations, CD103+ and CD103- LDC. Although accumulating evidence suggests that these LDC subsets play distinct roles in immune responses, their Immunity, Department functional role in immunity to infections remains largely unclear. Chlamydia is an intracellular bacterial of Medical pathogen that causes diseases of global public health concern. In this study, we have investigated to Microbiology, determine the role of CD103+ and CD103- LDC in protection against chlamydial lung infection. Methods: We examined the kinetics of LDC subsets following intranasal infection with C. muridarum University of Manitoba, (murine strain of C. trachomatis). We performed in vivo adoptive transfer experiments with CD103+ and Winnipeg, MB CD103- LDC to determine the ability of these LDC subsets in inducing protective immunity against challenge infection. Results: We found that both CD103+ and CD103- LDC expanded after C. muridarum infection. CD103+ LDC showed higher expression of CD40 and CD80, but not CD86, co-stimulatory molecules and increased IL-12 production compared with CD103- LDC. Adoptive transfer of CD103+ LDC isolated from C. muridarum infected mice conferred better protection than CD103- LDC against challenge infection. Cytokine analyses of the lung tissues and lung-draining lymph nodes from CD103+ LDC recipient mice revealed enhanced production of TH1 (IFN-γ) and TH17 (IL-17) cytokines compared to CD103- LDC recipients. Conclusion: Our results provide the direct in vivo evidence on the protective role of CD103+ LDC in host defense to an intracellular bacterial infection. In particular, these results have important implications for development of targeted prophylactic/therapeutic strategies against chlamydial infections. Structural Studies of the Aminoglycoside Modifying Enzyme APH(2")-IVa and Its Inhibitors Kun Shi and Albert M. Berghuis

Department of Bacterial resistance to antibiotic therapy presents a persistent threat to public health. Aminogly- cosides Biochemistry, McGill are a class of bactericidal antibiotics that interferes with bacterial ribosome function, leading to mistranslation of mRNA to yield defective proteins and thereby causing a detrimental effect on the mi- University, Montreal, croorganism. Clinical resistance to a number of aminoglycosides used against enterococci infections is QC conferred by members of the aminoglycoside-2″-phosphotransferase [aph(2″)] subfamily. Among these genes, aph(2″)-IVa has been characterized as a broad-spectrum drug-resistance gene in various enterococci species, widely known for their high level of endemic antibiotic resistance. In principle, if the resistance mechanisms are inhibited, the antibacterial activity of aminoglycosides will be restored. Having a detailed structural understanding of the aminoglycoside-modifying enzymes will thus help us in the design of either novel aminoglycosides immune to modification, or of specific inhibitors that can be co- administered with existing aminoglycosides. All known aminoglycoside phosphotransferases use either ATP or GTP as the phosphate donor in the phosphotransfer reaction, while their aminoglycoside substrate preferences are distinctly different. Members of the APH(3′) and APH(2″) subfamilies include some of the most prevalent resistance enzymes found in clinical isolates worldwide. During catalysis, APH(3′) enzymes are ATP-specific, whereas members of the APH(2″) subfamily are also able to use GTP. In this context, APH(2″)-IVa is particularly intriguing because of its nearly identical catalytic efficiencies with either ATP or GTP. This unique property presents the valuable opportunity of contrasting ATP versus GTP binding at the same active site and elucidating key structural features that influence nucleotide specificity among APH enzymes. At this time, our studies have produced seven crystal structures of APH(2″)-IVa, rendering it one of the most extensively characterized aminoglycoside resistance enzyme. Two structures of APH(2″)- IVa in complex with different aminoglycosides have shed light on the antibiotic binding mode and have shown that conformational changes occur upon substrate binding. Comparisons with the aminoglycoside binding site at the ribosome revealed important differences, which can be exploited in the design of next-generation aminoglycosides. In addition, crystal structures of APH(2″)-IVa in complex with ATP and GTP analogs, supplemented by steady state kinetics and mutational studies, have allowed us to rationalize the structural basis for dual nucleotide specificity. Comparative structural analyses with other APHs of known structures led us to identify key structural elements that influence ATP versus GTP selectivity among different subfamilies of APH enzymes. Equipped with this structural knowledge of the enzyme’s active site, we are currently striving towards identifying novel inhibitors for APH enzymes. Our focus is on small molecule inhibitors of divergent chemical scaffolds that have been developed against the kinase CK2, which is unique among eukaryotic protein kinases for its ability to utilize both ATP and GTP. Our crystal structures have shown strong parallels in the active site architecture between APH(2″)-IVa and CK2, and we believe that compounds which take advantage of the GTP-binding capability of CK2 can serve as a good starting point for the development of inhibitors active against APH(2″) enzymes. Given the range and diversity of aminoglycoside phosphotransferases, it is not surprising that inhibitors known to date are only active against a subset of APH enzymes, namely those that use ATP as the phosphate donor. We focus on the aminoglycoside phosphotransferases with GTP- binding capability, with the goal of discovering novel chemical scaffolds that can inhibit this clinically important family of resistance enzymes with high potency and specificity. Such compounds could serve as leads towards the development of adjuvants used in combination therapy with existing aminoglycosides. The Death Gene BNIP3 Regulates Excessive Mitophagy by Interacting with LC3 in Delayed Neuronal Death in Stroke Ruoyang Shi, Shenghua Zhu and Jiming Kong

Department of Human Introduction: Physiological mitophagy is essential for the mitochondrial quality control and homeostasis, Anatomy and Cell and is beneficial to neuronal survival in ischemic stroke. However, the excessive mitophagy has been proposed to contribute to the delayed neuronal death in stroke in recent studies. In this research, Science, University of we show that the mitochondria-interacting protein BNIP3 is an important upstream regulator of this Manitoba, Winnipeg, process. Specifically, BNIP3 could effectively interact with LC3 to trigger the excessive mitophagy and the MB subsequent neuronal damage. Methods: Immunocytochemistry, western blot, cell transfection, co-immunoprecipitation and ELISA were performed on in vitro and in vivo stroke models. Both BNIP3 wild-type (WT) and knock-out (KO) transgenic mice were used for tissue collection. Results: In primary neuronal cultures exposed to OGD and Reperfusion, the death-inducing BNIP3 was highly expressed and the time course and expression levels of mitophagy-related proteins (i.e. LC3, Beclin-1, and LAMP-2) were positively regulated by BNIP3. A strong direct interaction between mitochondria-located BNIP3 and the autophagosome marker, LC3, was detected by co-ip, immunocytochemistry and further quantified by ELISA, indicating BNIP3 may serve as an effective binding target on damaged mitochondria for the LC3 localization. By using western blot, we found the mitochondrial marker proteins (i.e. COXIV, TOMM22, and VDAC1) were well preserved in BNIP3 KO tissues compared to the WT tissues upon ischemia/hypoxia (I/H), and the infarct volume of ischemic brains was significantly reduced in BNIP3 KO mice compared to WT mice after 3-7 days recovery, demonstrating BNIP3 promotes an excessive elimination of mitochondria and contribute to the subsequent neuronal damage. Promoting or inhibiting mitophagy activities by using specific inducer or inhibitor didn't affect the expression patterns of BNIP3. Thus, BNIP3 is an upstream regulator of the excessive mitophagy in I/H-challenged neurons. Conclusions: Our results proved for the first time that BNIP3 could directly interact with LC3 and trigger the excessive mitophagy in delayed neuronal death in stroke. Global Alcohol Exposure Estimates by Country, Territory and Region for 2005 – A Contribution to the Comparative Risk Assessment for the 2010 Global Burden of Disease Study Kevin Shield

Institute of Medical Background: In 2004 alcohol consumption was the eighth leading risk factor for mortality, and the third Science, University of leading risk factor of death and disability was the third leading risk factor of death and disability. Reliable health data and statistics on alcohol exposure are required to formulate health policies and strategies. Toronto, Toronto, ON Purpose: Accordingly this study aimed to estimate the prevalence of lifetime abstainers, former drinkers and current drinkers, adult per-capita consumption of alcohol and pattern of drinking scores, by country and Global Burden of Disease region for 2005, and to forecast these indicators for 2010. Hypothesis: Unhealthy consumption of alcohol in terms of the way in which alcohol is consumed and the amount of alcohol consumed is prevalent in every region of the world in 2005 and 2010. Methods: Statistical modelling of alcohol consumption and alcohol drinking patters for 241 countries and territories were based on survey data and routine statistics. Per-capita consumption data were obtained with the help of the World Health Organization’s Global Information System on Alcohol and Health. Drinking status data were obtained from Gender, Alcohol and Culture: An International Study, the STEPwise approach to Surveillance study, the World Health Survey/Multi-Country Study and other surveys. Consumption and drinking status data were triangulated to estimate alcohol consumption across multiple categories. Results: In 2005 adult per-capita annual consumption of alcohol was 6.1 litres, with 28.8% stemming from unrecorded consumption; 17.1 litres of alcohol were consumed per drinker, 45.8% of all adults were lifetime abstainers, 13.6% were former drinkers and 40.6% were current drinkers. Lifetime abstention was most prevalent in North Africa/Middle East and South Asia. Eastern Europe and Southern sub-Saharan Africa had the most detrimental pattern of drinking scores, while drinkers in Europe (Eastern and Central) and sub- Saharan Africa (Southern and West) consumed the most alcohol. Conclusions: Just over 40% of the world’s adult population consumes alcohol and the average consumption per drinker is 17.1 litres per year. The prevalence of abstention, level of alcohol consumption and patterns of drinking vary widely across regions of the world; however, in every region of the world on average drinkers consume alcohol in a harmful manner. Combined Clinical and Cytogenetic Prognostic Markers Greatly Enhances Risk- Stratification of Medulloblastoma: A Global Community Effort David J.H. Shih1,2, Paul A. Northcott3, Marc Remke1,2, Andrey Korshunov4, David T.W. Jones3, Marcel Kool3, Stefan M. Pfister3,5, and Michael D. Taylor1,2,6, on behalf of the Medulloblastoma Advanced Genomics International Consortium (M.A.G.I.C.)

1Department of Laboratory Introduction: Medulloblastoma is the most common malignant pediatric brain tumour, and it comprises Medicine and Pathobiology, four molecular subgroups – WNT, SHH, Group3, and Group4 – which have disparate biological, clinical, and University of Toronto, demographic characteristics. Current treatment regimens stratify all medulloblastoma patients into risk Toronto, ON, 2Develop- groups based on clinical features including patient age, tumour histology, and metastatic stage. Recent mental and Stem Cell reports of the stark genetic differences among the four subgroups of medulloblastoma suggest that Biology Program, The subgroup-specific biomarkers may improve the risk-stratification of patients and help minimize adverse Hospital for Sick Children, treatment-related side-effects. 3 Division of Pediatric Patients and Methods: Molecular biomarkers for risk-stratification were identified from a copy-number Neurooncology, German profiling screen by single nucleotide polymorphism arrays on a discovery set (n = 629), consisting of Cancer Research Center samples acquired retrospectively from 43 cities around the globe. Tissue microarray analyses for select (DKFZ), Heidelberg, biomarkers were performed in an independent validation set (n = 453). Combined risk-stratification models 4 Germany, CCU were designed based on clinical and cytogenetic biomarkers identified by multivariate Cox proportional Neuropathology, German hazard analyses on the discovery set, and the models were subsequently tested on the validation set. Cancer Research Center Results: Subgroup status was strongly predictive of patient survival; it improved the predictive accuracy (DKFZ), Heidelberg, of a multivariate survival model on top of known clinical biomarkers. WNT patients had good survival Germany, 5Department of irrespective of clinical biomarkers. With the exception of metastatic stage, most clinical biomarkers were Pediatric Oncology, differentially associated with survival across the remaining subgroups. Similarly, most known cytogenetic University Hospital biomarkers were only prognostic within specific subgroups (e.g. MYC and MYCN amplifications). Our clinical- Heidelberg, Heidelberg, cytogenetic risk schema can identify low-risk (almost Wnt-like) Group3 patients, and high-risk (almost Germany, 6Department of Group3-like) SHH patients using only tools available in a modern neuropathology laboratory. Surgery, Division of Conclusions: Molecular subgroups and genomic aberrations in medulloblastoma are powerful biomarkers Neurosurgery and Labatt of prognostic outcome. Combining subgroup and cytogenetic biomarkers with established clinical Brain Tumour Research risk factors can substantially improve survival prediction, even in the context of heterogeneous clinical Centre, The Hospital for Sick Children, Toronto, ON treatments. Critically, the prognostic significances of biomarkers are often restricted to specific subgroups. By examining >1000 patients, we have identified several novel cytogenetic markers that identify very high risk, and very low risk groups of patients. The identified cytogenetic biomarkers can serve as high-priority candidates for prospective multicentre trials. Palmitoylation of G-protein Alphaq in Hypoxic Pulmonary H ypertension Anurag Singh Sikarwar, Martha Hinton, Prashen Chelikani, Shyamala Dakshinamurti

Department of Introduction: Hyperreactivity of thromboxane prostanoind receptor (TP) is a major contributor to Physiology and persistent pulmonary hypertension of the newborn (PPHN). We previously reported that in hypoxic pulmonary artery smooth muscle cells (PASMCs) TP couples mainly with G-protein alphaq (Gαq), which Pediatrics, Manitoba undergoes palmitoylation. Palmitoylation is a dynamic, reversible post-translational modification of Institute of Child the N-terminal cysteine residues of Gαq, which is required for efficient receptor-Gαq interaction. Health, Department of Objective: To study the role of Gαq palmitoylation in hypoxic pulmonary hypertension. Methods: PPHN was induced in newborn piglets by exposure to normobaric hypoxia (Fi02 0.10) for Oral Biology, University 72h; age-matched normoxic piglets served as controls. Serum deprived primary cultured myocytes of Manitoba, from newborn pig (day 0) were placed in hypoxic (10% 02) or normoxic (21% 02) environment for Winnipeg, MB 72h. Palmitoylable cysteine to alanine mutants of Gαq were obtained commercially and transiently transfected in HEK293T cells stably expressing TP. Calcium mobilization to TP stimulation was studied by calcium indicator dyes in hypoxic and normoxic conditions. Metabolic labeling of Gαq by 3H palmitic acid is currently being pursued under hypoxia and normoxia. Results: Pretreatment with palmitoylation inhibitor 2-bromopalmitate (2-BrP) decreased the maximum contractile response and calcium mobilization of hypoxic pulmonary artery rings and PASMCs respectively. 2-BrP had minimal effect on rings and PASMCs from normoxic piglets. Conclusions: Palmitoylation of Gαq could play role in hypoxia-induced hyperreactivity of TP. Proteomic Host-Response in H5N1 Influenza Virus Infections Philippe Simon, Patrick Chong, Stuart McCorrister, Garrett Westmacott and Darwyn Kobasa

Department of Medical Highly pathogenic avian influenza (HPAI) viruses are considered an important threat to global health. Microbiology, In avian species, these viruses have very high transmission and fatality rates. They are an agricultural and economical problem to poultry farms in South-East Asia and are propagated by migratory birds. University of Manitoba, Accordingly, their range includes the Eurasian continent and Africa. In humans, HPAI transmit poorly yet Winnipeg, MB, Public cause a very aggressive disease. Approximately 60% of infected patients will succumb to a fatal Acute Health Agency of Respiratory Distress Syndrome (ARDS). As in previous influenza pandemics, it is feared that HPAI may acquire high human-to-human transmission capacities either by mutation or through reassortment Canada, Winnipeg, MB of avian-human viruses. Two recent studies in ferrets have sparked an international controversy and demonstrated that only minimal point mutations are sufficient to sustain droplet transmission of H5N1 subtyped HPAI. An easily transmissible H5N1 virus could cause an aggressive disease for which we have limited countermeasures and no preexisting immunity. Very few comprehensive proteomic studies have been carried-out to understand the cellular events triggered by H5N1 infection in cells. Our goal is to better understand the proteomic response to HPAI infection and use this to derive new therapeutic strategies. We are currently using the iTRAQ labeling technique and infecting stable lung carcinoma (A549) and normal human bronchial epithelial (NHBE) cells with HPAI as well as low pathogenicity avian influenza (LPAI) and seasonal influenza viruses. This provides us with quantitative proteomic data on the unique host-response to HPAI H5N1 infection. By studying the early cellular events triggered by infection and comparing the protein dysregulation following infection with HPAI we hope to further our understanding of the cellular mechanisms involved in viral replication. This would give us critical information to develop new host-based therapies. Electrical Stimulation and Augmented Peripheral Nerve Regeneration in Diabetics: Potential Role of PI3-K Signaling Pathway B. Singh1, A. Krishnan1, V. Singh1, K. Koshy1, I. Micu1, D. Koshy1, C. Dalton2, D.W. Zochodne1

1Department of Introduction: Peripheral nerve regeneration is compromised in diabetes mellitus. Despite several Clinical mechanisms of neuroregenerative failure in diabetics have been proposed so far, unfortunately regenerative outcomes are still not promising. A short extracellular electrical stimulation (ES) paradigm Neurosciences and applied to nondiabetic nerves immediately following nerve injury and surgical repair enhances axon the Hotchkiss Brain regeneration and associated functional recovery. The potential impact of this intervention in diabetic Institute, University of nerves is uncertain. Methods: A cohort of type I diabetic (STZ induced) mice was maintained for 2 months, then divided Calgary, Calgary, AB into ES and sham stimulation groups following sciatic nerve crush. ES (20Hz, 3V, 0.1 ms, 1h) or sham stimulation was applied to the proximal injured sciatic nerves immediately after injury. Multifiber motor and sensory recordings and sensory behavioural testing were carried out on day 0 (before) and 14 and 28 days post nerve injury and repair. Skin and nerve tissues were processed at termination for intraepidermal nerve fiber density (IENFD) and myelinated axon counts respectively. In separate experiments in vitro, neurite outgrowth of isolated adult diabetic sensory neurons was measured following an ES paradigm (200mV, 20Hz, I h) applied through a multi-electrode array. Results: Diabetic mice were hyperglycemic and gained less weight than nondiabetic wild type controls. ES improved compound muscle action potential amplitudes and animals regained greater sensitivity to mechanical stimuli at 28 dpi. The intraepidermal nerve fibersreinnervating skin epidermis and density of regenerated tibial myelinated axons were higher with ES. In vitro, stimulated diabetic neurons showed higher neurite outgrowth than controls. In nondiabetic neurons, we also explored novel mechanisms of the benefits of ES. Isolated sensory neurons imaged by two photon microscopy demonstrated increases in intracellular calcium in response to ES. Following stimulation of nondiabetic nerves in vivo, PTEN expression was reduced, accompanied by rises in pAkt and ps6k levels in ipsilateral DRGs. PI3-K inhibitor but not Nic\2 blocked Neurite outgrowth. Conclusions: Overall, extracellular ES facilitates robust nerve regeneration in diabetic animals. The mechanisms may involve facilitation of PI3-K signaling by suppression of PTEN. Understanding the Link between Self-Determinance and the Mental Health of First Nations: A Qualitative Participatory Study J.E. Sjoblom and B. Elias

Department of Background: First Nations experience a disproportionate burden of mental health conditions, including Community Health significantly higher rates of suicide, depression, and substance use problems than the general population. Few studies have investigated the role of community level factors in predicting mental health outcomes of Sciences, Faculty of Aboriginal peoples in Canadian contexts. Medicine, University of Objective: To explore, qualitatively, the pathways through which community level factors may protect and Manitoba, Winnipeg, promote the mental health of community members in First Nations tribal areas in Manitoba, Canada. Methods: Study results from “The Mental Health of Tribal First Nations in Manitoba” report will be utilized MB to identify the tribal areas with the highest and lowest rates of mental health conditions. Two First Nations tribal areas, one with higher and one with lower rates of mental health conditions will be selected for in- depth qualitative study. The study will utilize a community-based, participatory research approach in which Tribal area Chief and Councils and their representatives will be engaged to negotiate consent, establish the communities’ mental health research priorities, identify key interviewees, determine preferred data collection methods, and ascertain knowledge dissemination protocol. Results: Not yet available. Significance: Understanding potentially protective effects of First Nation community level factors may provide valuable evidence to inform community-based mental health promotion and intervention initiatives. Moreover, a better understanding of how community level factors--such as self-governance and cultural continuity--protect and promote mental health could strengthen Aboriginal efforts to recover local control from proponents and from provincial and federal governments. Examining the Pathway to Diagnosis and Treatment of Lymphoma in Manitoba: Patient and System Related Factors Resulting in Delay Pamela Skrabek1,2, Matthew Seftel1, Alain Demers2,3, Brenda Elias2 and Donna Turner2,4

1Department of Medical Introduction: Lymphoma is the fifth most common cancer in Canada. Prompt diagnosis and treatment are Oncology and key requirements to improving patients’ survival and quality of life. Extended system delay, which is time from seeking medical attention to definitive diagnosis and treatment, is a concern. Haematology, Objectives: This study will examine system delay, starting from suspicion of cancer. Manitoba (MB) Health CancerCare Manitoba, (MH) and Cancer Registry (MCR) data will be analyzed to determine length of del ays and the influence Winnipeg, MB, of variables known to increase or decrease delay. How delay impacts survival in patients with aggressive lymphoma w ill also be determined. 2Department of Methods: We will create a cohort of MB adults, diagnosed with lymphoma between January 1, Community Health 2005 and December 31, 2010 using the MCR. A chart review will be conducted for a random subset of Sciences, University of patients (n= l 12) to establish a milestone framework. This will be compared to patient pathways built by linking the entire cohort with MH billing claims data, determining feasibility of constructing pathways Manitoba, Winnipeg, MB with this methodology. Multiple regression analysis will examine the influence of age, gender, lymphoma 3 Public Health, Agency subtype, residence area, contin uity of primary care, number/type of biopsies, diagnostic imaging delay and of Canada, Winnipeg, socioeconom ic status (SES) on length of system delay. Then, a survival analysis will examine how length of MB, 4Epidemiology and delay relates to survival controlling for stage, age, lymphoma subtype and gender. Anticipated Results: Given complexity in diagnosing lymphoma and the lack of a coordinated approach Cancer Registry, we expect system delay to exceed the Cancer Patient Journey Initiative (CPJI) goal of 60 days from CancerCare Manitoba, “suspicion” to treatment in most. More aggressive lymphomas are expected to have fewer delays. Measures Winnipeg, MB of health care access including rural residence, SES and lack of continuity of care are expected to increase delay. Much of the delay is expected to be prior to specialist appointment. Conclusion: Analysis of system delay in the patient pathway for lymphoma will guide policy in order to improve access and outcomes by establishing baseline length of delay and possible reasons for in appropriate delay. The CPJI aims to improve efficiency and experience of Manitobansdiagnosed with cancer, change must be informed by accurate, population based data. Electrophysiological Properties of Dystrophin-deficient Purkinje Neurons Wanda M. Snow, Judy E. Anderson, Mark Fry

Department of Introduction: Dystrophin protein, abundant in muscle, also resides in cerebellar Purkinje neurons. Its Psychology, University absence causes Duchenne muscular dystrophy (DMD) in humans, a fatal X-linked genetic disorder characterized by muscle wasting and cognitive deficits. Dystrophin plays a role in ion channel and GABA of Manitoba, Winnipeg, receptor maintenance. As dystrophin deficiency results in altered ligand-gated ion channel function, and MB ion channel function dictates intrinsic neuronal membrane properties, an absence of dystrophin may perturb intrinsic electrophysiological properties of Purkinje neurons. We previously noted a 30% increase in dystrophin-puncta density in Purkinje neurons from the lateral cerebellum, implicated in cognition, compared to vermal Purkinje neurons, involved in motor function, prompting an examination of Purkinje neuron electrophysiology between functional regions in the normal and dystrophin-deficient cerebellum. Methods: Purkinje neurons were dissociated from vermal and lateral cerebellum in C57BL10 and mdx mice, a genetic model of DMD, and subjected to whole-cell patch clamping. Electrophysiological parameters, including spontaneous action potential frequency and evoked waveform properties (i.e. threshold, duration, amplitude), were studied under current clamp mode and analyzed using a 2x2 (Region x Genotype) ANOVA. Results: Patch clamp recordings revealed decreased firing frequency in vermal (9.53 ± 4.80 Hz) vs. lateral (38.05 ± 8.31 Hz) Purkinje neurons in control mice (p<0.002). In the lateral cerebellum, dystrophin deficiency resulted in decreased firing frequency in Purkinje neurons mdx( : 12.36 ± 5.74 Hz; controls: 38.05 ± 8.31 Hz, p=0.003). The amplitude of evoked action potentials was greater in Purkinje neurons from the vermal (92.74 ± 1.67 mV) vs. the lateral (85.00 ± 2.00 mV; p=0.004) region, irrespective of genotype. Action potentials were shorter in duration in vermal (0.42 ± 0.01 msec) than lateral (0.47 ± 0.02 msec) Purkinje neurons (p<0.038). Conclusions: Purkinje neuron action potential waveform parameters differed based on cerebellar region, as did the rate of spontaneous activity. Dystrophin deficiency reduced spontaneous activity in Purkinje neurons from the lateral region, associated with cognition. These findings highlight the importance of distinguishing cerebellar functional zones in characterizing Purkinje neuron electrophysiology and suggest that cognitive deficits associated with DMD may be mediated by the loss of dystrophin due to dysregulation of intrinsic membrane properties. Differential Effects of Lipophilic Versus Hydrophilic Statins on Survival of Human Atrial Fibroblasts in vitro K.K. Sran, Y. Li, S. Ghavami, R.C. Arora, L.A. Kirshenbaum, D. Freed

Institute of Cardiovascular diseases (CVD) leading to heart failure are associated with varying degrees of myocardial cell Cardiovascular loss and cardiac fibrosis. Cardiac fibrosis is prevalent in conditions such as myocardial infarction, ventricular hypertrophy due to hypertension or valvular heart disease, or diabetic cardiomyopathy, and has limited Sciences, Department therapeutic options for its treatment. HMG-CoA Reductase (HMGR) inhibitors ("statins") have been shown of Physiology, to limit cardiovascular events in patients with CVD through modulation of lipid profile by downregulating University of Manitoba, production of cholesterol precursor L-mevalonate. Mevalonate depletion has been shown to induce autophagy, apoptosis, and endoplasmic reticulum stress in various cell types. However it is unclear if this is a Winnipeg, MB class effect or a phenomenon specific to certain compounds. We hypothesize that lipophilic statins induce cell death in primary human atrial fibroblasts (hATF) via mevaloate depletion; whereas hydrophilic statins do not have the same effect on the hATF viability. The protocol received IRB approval, and hATF were isolated from the atrial appendage collected from consented patients undergoing open heart surgery and cultured in standard conditions. Cells were treated with atorvastatin, simvastatin (lipophilic statins) or pravastatin (hydrophilic statin) 0.1, 1.0, 10uM for 24, 48, 72, or 96 hours. Cell viability was assessed using an optical MTT assay. Expression of key apoptotic and autophagy protein markers was assessed by western blot technique. Rescue of viability was achieved through coincubation with mevalonate. hATF treated with 0.1-10uM atrovastatin or simvastatin showed progressively reduced cell viability in a time and dose dependent manner which was associated with expression of key apoptotic cascade proteins Bcl2, Bax and cleaved caspase 3, showing a clear induction of apoptosis. Treated hATF also showed an increase in Atg5-12 expression at 24 hour, indicating as early autophagic response. Viability could be rescued by coincubation with mevalonate. Treatment with pravastatin did not affect the cell viability or expression of autophagic or apoptotic markers. We conclude that statins induce cell death in hATF, which is mediated by mevalonate depletion through HMGR inhibition. Lipophilic statins impair the viability of primary hATF in culture by induction of intrinsic apoptotic pathways whereas hydrophilic statins do not affect hATF viability in vitro. This could be due to the hydrophilic characteristic of the molecule, preventing its uptake by the cell. This may represent an additional pleiotropic effect of statins. Understanding the Regulation of LAG-3 Expression in Healthy and HIV Infected People A. Stalker1, J. Juno1, K.R. Fowke1,2,3

1Department of Background: Regulation of the immune system involves the expression of immune inhibitory markers, Medical Microbiology, including LAG-3, PD-1, Tim-3 and 2B4, which inhibit T cell effector functions limiting inflammation. During chronic infections, including HIV infection, the inappropriate expression of inhibitory markers contributes University of Manitoba, to a dysfunctional state known as immune exhaustion. Although many exhaustion markers are upregulated 2Department of during HIV infection, LAG-3 expression is consistently low on CD4+ and CD8+ T cells. Currently, little is Community Health known about the function of LAG-3 on non-T cell lymphocytes, and the factors regulating LAG-3 expression are not well defined in humans. Preliminary data from our lab suggests that LAG-3 is strongly expressed Sciences, University of on natural killer (NK) cells and invariant natural killer T (iNKT) cell subsets and can be expressed within 6hrs Manitoba, Winnipeg, post stimulation. Murine studies have observed production of soluble LAG-3 (sLAG-3) by metalloprotease MB, 3University of cleavage of surface protein. Human metalloprotease activity on surface LAG-3 has not been studied and the role of sLAG-3 is unknown. Nairobi, Nairobi, Kenya Hypothesis: Based on these data, we hypothesize that LAG-3 is rapidly up-regulated from an intracellular store to the surface of activated T cells and iNKT cells. We also hypothesize that sLAG-3 will be generated through both the expression of splice variant mRNA and metalloprotease cleavage of surface LAG-3 and that sLAG-3 will function to inhibit T cell activation. The removal of surface LAG-3 by metalloprotease cleavage will prevent sustained expression during chronic HIV infection. Methods: We will test this hypothesis using flow cytometry to measure surface and intracellular LAG-3 and ELISA to measure soluble LAG-3. Significance: Improved understanding of LAG-3 may contribute to treatment options for chronic infections and HIV prevention through regulation of immune activation. Reduced Interference Between Identity and Expression Processing With Dynamic Faces Brenda M. Stoesz and Lorna S. Jakobson

Department of Facial motion cues facilitate identity and expression processing (Pilz et al., 2006). To explore the possible Psychology, University basis of this dynamic advantage, we used Garner’s selective attention paradigm (Garner, 1976) to determine whether adding dynamic cues alters the way that identity and expression processing interact, and whether of Manitoba, Winnipeg, this varies depending on the age of the viewer. Adults (ages 18-26), adolescents (ages 12-13), and children MB (ages 6-7) made speeded judgments of the expression (or identity) of static and dynamic faces while identity (or expression) was either held constant (baseline block) or varied (orthogonal block). Accuracy was high (>90%) for all three groups. We calculated corrected Garner interference scores by determining the percent change from baseline RT seen in the orthogonal block. A 2 (Task: Expression, Identity) X 2 (Mode: Static, Dynamic) X 3 (Age Group: Adult, Adolescent, Child) ANOVA conducted on these scores revealed that interference was stronger in the Expression than in the Identity task, and with static compared to dynamic stimuli. However, follow-up tests conducted on the significant Task X Mode interaction showed that, while the addition of dynamic cues led to a significant reduction in Garner interference for both tasks, the effect was much more dramatic for the Expression task. Although young children took significantly longer than adolescents or adults to make their judgments, no age-related differences in Garner interference were observed. Reductions in interference after the introduction of dynamic cues might arise if viewers focus more selectively on specific facial features, or if they integrate multiple cues more effectively, when viewing moving as opposed to static faces. Both of these ideas hold merit and, indeed, it is possible that individual differences in processing style determine which strategy a viewer will adopt. These results highlight the importance of using naturalistic, dynamic stimuli in studies of face processing. Treatment Decision Making Among Older Men and Women with Colorectal Cancer Fay J. Strohschein

Ingram School of Purpose: To gain in-depth understanding of cancer treatment decision making (TDM) from the perspective Nursing, McGill of older men and women with colorectal cancer (CRC) in the first 3 to 4 months following diagnosis. Specific aims include identification of age-related and gender-related individual and contextual factors that University, Montreal, influence TDM. QC Significance: In Canada, 43% of new cancer cases and 61% of cancer deaths occur in people 70 years and older. Older adults with cancer present a particular set of needs and concerns that are often under- recognized, remaining unmet and under-studied. Given that the number of older Canadians is expected to more than double within 25 years, the need to address issues related to cancer care in this population is paramount. CRC is a particular concern among older adults. More than half of all CRC diagnoses and deaths in Canada occur in those aged 70 or older, and CRC is the second most common cause of cancer incidence and mortality among these older Canadians. TDM relating to surgery, radiotherapy, and/or chemotherapy is an ongoing part of the CRC trajectory. Variations in health/functional status, biological aspects of cancer/aging, changes in social support networks, and increased tension between quality and quantity of life present unique challenges to cancer TDM for older adults. Clinical practice guidelines and recommendations, as well as studies suggesting under treatment of older adults with CRC, recognize the important role of patient preference in TDM; however, little is known about the process of TDM from the perspective of older adults themselves. Design: Constructivist grounded theory methodology, with theoretical underpinnings in symbolic interactionism, will guide study design. Symbolic interactionism suggests human behavior is based on meaning derived through social interaction and internal interpretive processes. Setting: An urban university- affiliated hospital serving a diverse population. Sample: Participants will include adults 70 years and older, recently diagnosed with stage II or III CRC, who have not yet started treatment. Theoretical sampling will be used to choose participants who are expected to contribute data that elaborate and refine emerging theoretical categories. Methods: In‐depth interviews exploring participant perspectives of TDM will be conducted before treatment begins and again 3‐4 months later. Between interviews, participants will be asked to record thoughts and experiences related to TDM in a provided journal. Journal entries and emerging conceptualizations will guide the second interview. Medical and demographic questionnaires will allow for detailed description of participants. Analysis will occur concurrently with data collection and will be guided by the principles of constant comparison and questioning, involving methods of coding and memo writing, using NVivo 10 software. Implications: Health care professionals play an important role in helping patients navigate treatment decisions, and in advocating for patients in health services and policy development. In-depth understanding of TDM is needed to optimize treatment outcomes, inform development of supportive interventions, and mobilize appropriate health services for older adults with CRC and their families. The substantive theory developed in this study will provide a framework to inform future research and practice. FRIZZLED-7 is a NOTCH3 Specific Target in Human Mammary Epithelial Cells Yu Jia Sun, Afshin Raouf

Cancer Care Manitoba, Objectives: The Notch signaling cascade is a highly conserved signaling pathway that has demonstrated Department of roles in the normal mammary gland development and breast carcinogenesis. The current paradigm of Notch signaling indicates that upon ligand binding each of the four NOTCH receptors (NRs) indiscriminately Immunology, regulate the transcription of same set of target genes. Hence, NRs are thought to have much overlap in their University of Manitoba, biological functions. Recent evidence however, indicates that signaling through NR3 alone is essential for Winnipeg, MB maintaining the luminal cell differentiation potential of the bipotential human breast epithelial progenitors. This observation suggests that NRs play non-redundant roles in regulating the differentiation of breast epithelial cells and that they may have unique target genes. The objective of this project is to identify NOTCH3 specific target genes in regulating luminal differentiation. Methods and Results: Lenti viral transduction was used to knockdown the transcript levels of each NRs via shRNA technology, the RNA transcript levels of each NR and Notch target genes were assessed through qPCR array; the protein levels were assessed through Western blots and/or FACS. The qPCR array identified five genes whose transcript levels were altered only with decreases NR3 protein and RNA levels. A subset of three genes that have higher expression in luminal progenitors (where NR3 was the highest) were chosen as potential NR3 specific targets. Their differential expression was further validated using independent sets of qPCR primers. We found that a decrease in NR3 transcripts only but not other NRs lead to decreased FRIZZED-7 (FZD7) transcripts and protein levels. Interestingly, forced expression of activated NR3 but not NR1 or NR4 lead to increased transcript and protein levels of FZD7. Further more, we found that FZD7 has the same expression profile as NOTCH3 in subsets of primary mammary epithelial cells. Conclusion: Our data shows that FZD7 is a previously unknown unique target of NR3. Since Notch receptors are potent mammary oncogenes, understanding their precise mechanisms of action provides an insight into how their altered functions can lead to breast cancer and/or contribute to pre-malignant lesions. Our data points to a crosstalk between the Notch signaling and the Wnt signaling pathways that may be responsible for luminal cell differentiation during the human breast development. Effect of Surface Charge and Magnetic Field on Iron Oxide Nanoparticle Permeability in a Cell Culture Model of the Blood Brain Barrier (BBB) Zhizhi Sun, Matthew Worden, Vinith Yathindranath, Torsten Hegmann, Donald Miller

Department of Surface charge and applied magnetic fields affect cellular uptake of iron oxide nanoparticles (IONPs). Pharmacology and However, their effects on permeability of IONPs across the BBB remain unexplored. The aim of this study was to determine the impact of IONP surface charge on permeability in a cell culture model of the BBB in Therapeutics, the presence and absence of a magnetic field. Permeability of positively (AmS-) and negatively charged University of Manitoba, (EDT)-IONPs was evaluated in confluent bEnd.3 monolayers grown on polycarbonate membrane inserts Winnipeg, MB (3 µm pore). Our results show neither IONPs were not transported across the monolayer even in presence of magnetic field. When tight junctions were disrupted using D-mannitol, 44% and 28% of EDT-IONPs were found across the monolayer in presence and absence of magnetic field, respectively. Under the same conditions, there was only 10% permeability of the AmS-IONPs detected. In conclusion, negatively charged IONPs have a more favorable permeability profile in brain endothelial cells via paracellular route following osmotic disruption. Use of EDT-IONPs during transient disruption of BBB, may improve nanoparticle based drug delivery to the brain. A Whole-Genome RNA Interference Screen Identified Human Cellular Factors which Affect the Replication of Myxoma Virus Wondimagegnehu M. Teferi, Kristopher Dodd, Rob Maranchuk, Nicole Favis, David H. Evans

Department of Medical Introduction: Myxoma virus (MYXV), a member of the family of poxviruses, naturally infects only Microbiology and rabbits. However, recent studies showed that the virus has an oncolytic potential due to its ability to selectively infect and kill human cancer cells both in culture and in xenograft tumor models in mice. Immunology and Li Ka The factors which enable the virus to cross the species barrier are not clearly known. Moreover, how Shing Institute of various cellular factors in general affect the replication of poxviruses is not well understood. We conducted Virology, University of a human whole-genome RNA interference (RNAi) screen to identify and characterize cellular factors which affect the replication of MYXV in cancer cells. Alberta, Edmonton, AB Methods: RNAi screens were performed by using human whole-genome (25,585 genes), kinome (986 genes) and custom-verification (88 genes) siRNA libraries. MDA MB-231 cells, a human breast cancer cell line, were transfected with siRNAs for 72h and then infected with β-galactosidase expressing MYXV for 48h. Following lysis of the cells, β-galactosidase assay was performed as a surrogate marker for virus replication. Toxicity screens were also performed using alamarBiue assay. Data analysis was carried out using the DAVID and PANTHER algorithms. Finally, independent experiments were used to verify some of the hits. Results: The screens identified 711 and 333 siRNAs which increased and decreased the replication of MYXV, respectively. A screen performed with a custom-verificationlibrary containing siRNAs which target some of the hits of the screens predicted 25% true positivity. Functional and pathway classification of the hits identified a number of statistically significant pathway and functional classes such as glycolysis, the cell cycle control system and the MAPK pathway. Conclusion: The findings of these experiments help to understand how various cellular factors affect the replication of MYXV in particular and poxviruses in general and how these factors can be manipulated to harness the oncolytic potential of MYXV. The Development and Optimization of Image-Based Screening Approaches for the Identification of Novel Human Chromosome Instability Genes Laura L. Thompson

Manitoba Institute of Introduction: Colorectal cancer has a significant impact on the lives of Canadians and is the second Cell Biology, University leading cause of cancer related deaths in Canada. Last year approximately 9,200 Canadians lost their lives to colorectal cancer, while 23,300 new colorectal cancer diagnoses were made. Approximately 85% of these of Manitoba, Winnipeg, colorectal tumors exhibit chromosome instability (CIN) which is defined as an increase in the rate at which MB whole chromosomes or large chromosomal fragments are gained or lost. CIN is associated with highly aggressive tumors, poor patient prognosis and the acquisition of multi-drug resistance, yet the genes and biological mechanisms responsible for regulating chromosome stability in humans remain largely unknown. Methods: In order to identify novel CIN genes we have begun to develop and optimize two high- throughput image-based screening platforms. We have generated a DsRed-LacI fusion gene that has been transduced into a modified HT-1080 cell line harbouring lac operator repeats at 11q13. Specific binding of DsRed-LacI to the lac operators allows for visualization of 2 fluorescent foci (1 focus/chromosome) within each nucleus. During the screen, human candidate genes identified through cross-species approaches will be silenced using siRNA techniques, and CIN will be monitored as a change in the expected number of nuclear foci. In addition to the fluorescence-based screen, nuclear volume will be monitored as changes in chromosome content are expected to correlate with changes in nuclear size. A subset of the putative human CIN genes identified by these image-based screens will be subjected to biochemical and cytogenetic analyses in order to validate them as CIN genes. Results: We have successfully generated cell lines that stably express fluorescently-tagged LacI. The DsRed fluorescent label was specifically selected during the optimization process as it produced more distinct nuclear foci. The J21 cell line was selected over another modified HT-1080 cell line, as it more consistently showed two nuclear foci. Preliminary imaging data suggests that both screening methods will effectively detect CIN and will facilitate the identification of human CIN genes. Conclusion: The identification of human CIN genes will contribute to a greater understanding of fundamental biology, and provide insight into the tumorigenic process. Combination of Micro-CT and a Novel Vascular Contrast Agent as a Pre-Clinical Imaging Technique to Characterize the Vasculature near Bone Justin J. Tse1,2,3, Joy Buyze-Dunmore2, Maria Drangova1,2,3, David W. Holdsworth1,2,3

1Department of Introduction: Osteoarthritis (OA) is a growing concern for many aging individuals. Many questions Medical Biophysics, remain in the initiation and progression of this joint disease. A leading hypothesis suggests that vascular deficiencies near bone results in decreased nutrient and oxygen flow to the bone, and a reduction in waste Western University, removal from the bone, leading to the cartilage and bone loss associated with OA. Thus, studying vascular 2Imaging Research changes associated with bone will provide more information into the initiation and progression of OA. Laboratories, Robarts However, two challenges are faced with visualizing vasculature: very small size (5-10 um), and poor inherent contrast. Research Institute, Objective: We propose the combination of dual-energy micro-CT (DECT) and a novel contrast agent for the London, ON 3Schulich visualization of vasculature associated with bone. This combination will overcome vasculatures’ small size School of Medicine and poor inherent contrast. Materials and Methods: We propose lanthanide nanoparticles as a base for the contrast agent. Lanthanides and Dentistry, Western possess an absorption K-edge (50-60 keV) that falls within the energy range of micro-CT scanners (30-90 University, London, ON kVp). Additionally, nanoparticles will ensure filling of the smallest vessels (5-10 um). Vascular changes will be studied in post-mortem control and surgically induced OA rats. Sacrifice, perfusion with the novel contrast agent, and subsequent micro-CT scanning will be done 8-weeks post surgery. Results: Preliminary results have successfully demonstrated the novel contrast agent perfusin a mouse and providing sufficient vascular contrast. Currently, only single energy (90 kVp) micro-CT scans were performed. However, simple image thresholding enabled the nutrient artery that feeds and runs through the tibia to be clearly observed and differentiated from surrounding bone. Conclusion: The combination of a novel perfusion contrast agent in conjunction with DECT imaging will result in individual images of bone and vasculature, allowing vessel micro-architecture, patency or blockage, and density quantification. This information will provide more insight into the initiation and progression of OA. Type VI Secretion Systems Effectors Reflect Diversity Within the Species Vibrio Cholerae Daniel Unterweger and Stefan Pukatzki

Department of Medical Introduction: Vibrio cholerae is a Gram-negative bacterium found in the marine environment. Ingestion Microbiology and of virulent V. cholerae can cause cholera, a disease characterized by watery diarrhea that can be fatal if untreated. Approximately 25% of all sequenced Proteobacteria (including V. cholerae) encode a type Immunology, Faculty of VI secretion system (T6SS) to deliver toxins into prokaryotic and eukaryotic cells. The V. cholerae 037 Medicine and serogroup strain V52 employs three T6SS effectors that target the bacterial envelope of prey bacteria. Dentistry, University of V. cholerae V52 and other strains encode immunity proteins downstream of their cognate effector to protect themselves against a T6SS-mediated attack by bacteria of the same strain or other strains that Alberta, Edmonton, AB encode the same effectors. However, some V. cholerae strains are sensitive to T6SS-mediated killing by V. cholerae V52 and are subsequently outcompeted. This led to the hypothesis that V. cholerae encodes a variety of T6SS effector-immunity pairs. Methods: In each of 37 sequenced V. cholerae strains, T6SS effector sequences were identified, translated and aligned. Previously uncharacterized T6SS effectors were analyzed by PLASTB and HHPRED. Strains of different compatibility groups were mixed and incubated on an agar plate, then harvested and enumerated to determine the competitive index. Immunity genes were expressed in trans to test the switch of complementation groups. Results: This analysis revealed a diverse set of modular T6SS effectors within the species V. cholerae. Strains that encode the same effector-immunity pairs were divided into compatibility groups. Strains within a compatibility group were shown to coexist and are thus compatible whereas strains of different groups outcompete each other and are thus incompatible. Conclusion: This form of kin selection may permit the evolution of distinct lineages, including those that give rise to toxigenic strains. Effects Of Dextromethorphan Mediated Bitter Taste Receptor Activation In Pulmonary Artery Smooth Muscle Cells Jasbir Upadhyaya1, Nisha Singh1, Anurag S. Sikarwar2, Rajinder P. Bhullar1, Shyamala Dakshinamurti2, 3, Prashen Chelikani1,3

1Department of Oral Introduction: Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to Biology, 2Department muscle relaxation and bronchodilation. However, the presence of T2Rs in human pulmonary artery smooth muscle cells (hPASMCs) has not been established. The previous finding in airway smooth muscle led to our of Pediatrics, hypothesis that T2Rs, if present in hPASMCs, might be involved in regulating the vascular tone. Physiology, University Methods: The expression of T2Rs in PASMCs was revealed by performing PCR. Functional characterization of of Manitoba, Winnipeg, the expressed T2Rs was done by calcium imaging after stimulating PASMCs with bitter agonists. Previously in site-directed mutational studies, we have characterized the response of T2R1 to bitter compound MB, 3Manitoba dextromethorphan (DXM), therefore T2R1 was selected for further analysis in this study. Knockdown of T2R1 Institute of Child in PASMCs was done by transiently transfecting the cells with T2R1-specific shRNA using nucleofactor kit. Health, Winnipeg, The levels of endothelin-1, a biomarker of systemic hypertension, were measured by ELISA. Finally, to analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial rings MB were pursued. Results: RT-PCR analysis revealed the expression of multiple T2R transcripts in hPASMCs. Functional analysis showed that these cells responded to many bitter-tasting compounds, by increasing intracellular calcium concentration, indicating that T2Rs in hPASMCs are functional. Knockdown of T2R1 in PASMCs decreased mRNA levels and DXM-induced calcium responses by up to 40%. ELISA data obtained from in vitro studies using hPASMC showed that DXM-treatment led to increased levels of endothelin-1. Myographic study showed that incubation of porcine pulmonary arterial rings with DXM led to their contraction. In contrast, DXM treatment of porcine pulmonary airway rings led to muscle relaxation, which agrees with previously published results. Conclusion: This study suggests that stimulation of T2R-expressing pulmonary artery with DXM leads to vasoconstrictor responses. Longitudinal Study of CD3ζ-Chain and T Cell Functions in the Correlation of the Disease Status in Head and Neck Cancer Patients Deepak Upreti1, Man-li Zhang1, Elena Bykova1, Alok Pathak2 and Sam K.P. Kung1

1Department of Background: Despite advances in multi-modality treatments that involve surgery, radiation, and Immunology, chemotherapy targeting primarily tumor cells in clinical practices, the 5-year disease free survival of the Head and Neck patients has remained low at around 50% over the past 35 years. The follow-up protocols University of Manitoba, currently in practices therefore do not seem to diagnose treatment failures and recurrences early enough. 2Department of Earlier reports showed a decreased expression level of CD3ζ chain in T cells of Head and Neck cancer Surgery, University of patients in general, suggesting that this could be a potential biomarker of head and neck tumor. Rationale: The development of head and neck squamous cancer (HNSCC) is strongly influenced by Manitoba, Winnipeg, the host immune system. Among various immunosuppressive mechanisms reported in HNSCC, we and MB others observed a reduced CD3ζ expression in both tumor infiltrating lymphocytes and peripheral blood lymphocytes in cross-sectional studies. The CD3ζ-chain plays a key role in the TCR receptor assembly, surface expression and signaling in T cells. Its relevance as a prognostic biomarker of HNSCC however has not been evaluated in a longitudinal study. We hypothesized that CD3ζ-chain expression would be a useful biomarker that predicted prognosis and disease progression in HNSCC patients. Method and Results: Patients’ cohort is established at Cancer Care Manitoba. Peripheral blood is being collected at the time of diagnosis and at different time intervals ranging from 2 months to 2 years. Lymphocytes are being isolated from heparinized peripheral blood by Ficoll-Hypaque at each time point. They are stained for surface CD3, CD56, and intracellularly for CD3ζ expression. Trends in the serial levels of CD3ζ chain is being measured using flow cytometry. Cross-sectional analysis of 69 patients sample showed that there was a significant difference in the CD3ζ expression between healthy and patient samples. Our preliminary analyses showed also that (i) the percentages of T cells in the PBMC of Healthy and HNSCC patients were comparable( ~65 %) ; (ii) the CD3ζ expression in T cells was restored in many (8/10) of the follow-up patients in a longitudinal study( iii)there was no difference in the CD3ζ expression in the NK cells of the HNSCC patient and control (iv) the percentage of NK cells was higher in patients when compared to the healthy donor (v) Patients have significantly lower IFN-gamma level in terms of MFI when stimulated with viral peptides CEF as compare to controls which was correlated with reduced CD3 ζ-chain. Small Molecule Inhibition of the Glycosidase NagZ Suppresses Beta-Lactam Resistance in Pseudomonas aeruginosa Grishma Vadlamani1, Yves Blériot2, Brian L. Mark1

1Department of Introduction: AmpC β-Lactamase overproduction, a major cause of β-lactam antibiotic resistance in Microbiology, Pseudomonas aeruginosa, is exacerbated by the inactivation of dacB, encoding the non-essential penicillin- binding protein 4 (PBP4). The glycoside hydrolase NagZ mediates the formation of 1,6-anhydromuramoyl- University of Manitoba, peptide - a catabolite of peptidoglycan recycling and inducer of AmpC expression. Its pivotal role in Winnipeg, MB, 2UMPC regulating AmpC β-lactamase expression makes NagZ a promising target for small molecule inhibitors Univ Paris 06 designed to suppress β-lactam-resistance. Azepanes are seven-membered iminoalditols that have been shown to block glycosidase activity. This study characterizes the ability of MM-60, a substituted azepane, to enhance β-lactam susceptibility in P. aeruginosa PA∆dacB. Methods: Minimum inhibitory concentrations (MICs) were performed in triplicate by the broth microdilution method in a 96-well plate using appropriate serial dilutions of ceftazidime in 100 μl of cation-adjusted Mueller-Hinton medium. Addition of 50 μl MM-60 to a final concentration of 1.3 mM was followed by inoculation with 50 μl of ~104 cells taken from subcultures of P. aeruginosa ∆dacB grown to an optical density (OD600) of ~0.5. MICs were determined after incubation of plate for 18 h at 37 °C in a shaker incubator. Results: In the presence of 1.3 mM MM-60, the MIC of P. aeruginosa ∆dacB is reduced by half from 24 μg/ml to 12 μg/ml ceftazidime. Conclusion: Glycosidase inhibitors including substituted azepanes have been used in the treatment of disorders such as diabetes and influenza. The results of this study demonstrate the efficacy of this class of inhibitors in suppressing β-lactam resistance in P. aeruginosa as a consequence of NagZ inactivation, and provide further support for a treatment strategy based on co-administration of β-lactam antibiotics and small molecule inhibitors targeted towards NagZ. Rapid Suppression of Human Growth Hormone Gene Expression Following Excess Caloric Intake and Hyperinsulinemia can be Prevented by Physical Activity Hana Vakili, Yan Jin, Peter A. Cattini

Department of Introduction: Growth hormone (GH) is a major metabolic homeostatic factor, and its levels are decreased Physiology, Division of in obese individuals; however this decrease happens earlier in response to excess caloric (“food”) intake and excess insulin release (hyperinsulinemia) before obesity occurs. The underlying mechanism responsible Endocrinology and for this suppression of GH is not known. The aim of this study is to use a “humanized” transgenic mouse Metabolic Diseases, containing the intact human (h) GH gene locus (hGH/CS-TG), and assess the effect of a high caloric intake University of Manitoba, on hGH synthesis. We hypothesize that a high caloric intake will result in hyperinsulinemia, and lead to chromatin remodeling of the hGH gene promoter resulting in a decrease in hGH gene expression, which can Winnipeg, MB be prevented through increased energy expenditure (“exercise”). Methods: Four-week old male hGH/CS-TG mice were assigned to non-exercise and exercise (50 minutes swimming daily) groups and were fed either a high-fat (60 kcal%) or a low-fat (10 kcal%) diet for 3 days. Body weights and average food intake were recorded daily. Blood glucose (using a glucometer), insulin, C-peptide and hGH levels were assessed from serum by ELISA. Human GH gene expression (RNA) was assessed by quantitative PCR. Relative level of histones H3/H4 hyperacetylation and recruitment of RNA polymerase II at the hGH promoter, were assessed as an indication of promoter accessibility/transcriptional status in pituitary cells by chromatin immunoprecipitation assay. Results: High fat diet for 3 days created a state of excess insulin and C-peptide but not hyperglycemia, without weight gain. Metabolic changes during excess caloric intake were associated with a decrease in both hGH RNA levels and secretion into the circulation. This was associated with decreases in histones H3/H4 hyperacetylation and RNA polymerase II occupancy and, thus, accessibility of the hGH promoter. Introduction of “exercise” significantly muted the effect of excess caloric intake on insulin and C-peptide levels. There was also a corresponding blunting with exercise of the decrease in hGH promoter hyperacetylation and gene expression/RNA levels seen in the absence of prescribed physical activity. Conclusion: These studies indicate a link between caloric intake and physical activity on hGH gene regulation through a possible effect of excess insulin on hGH promoter structure and activity in pituitary cells in vivo. A better understanding of GH suppression as an early event in the development of obesity may provide valuable insights into the pathogenesis of obesity. Decreased Nuclear Receptor Activity and Epigenetic Modulation Mediates Down- Regulation of Drug Metabolizing Enzymes in Chronic Kidney Disease T.J. Velenosi1, D.A. Feere1, G. Sohi1, D.B. Hardy1,2, A. Fu1, B.L. Urquhart1,2

1School of Medicine Background: Patients with Chronic Kidney Disease (CKD) require many medications to treat their renal and Dentistry, failure and associated co-morbidities. CKD patients experience an increased incidence of adverse drug events compared to patients without renal disease. The majority of prescribed medications are metabolized Department of by the drug metabolizing enzymes CYP3A and CYP2C. These enzymes are regulated by the nuclear Physiology and receptors pregnane X receptor (PXR) and hepatic nuclear factor alpha (HNF4). CYP3A and CYP2C expression Pharmacology, is decreased in CKD; however the mechanism(s) by which this occurs is unknown. The objective of this study was to determine the mechanism of hepatic drug metabolizing enzyme down-regulation in CKD. We University of Western hypothesize that altered drug metabolism in CKD is secondary to decreased nuclear receptor binding and Ontario, London, ON epigenetic modulation in the CYP3A and CYP2C promoter. 2Lawson Health Methods: Hepatic CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic Research Institute, mediated differences in the promoter region of these enzymes. London, ON Results: Hepatic CYP3A2 and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P<0.05). RNA polymerase II binding to the CYP3A2 and CYP2C11 promoter regions was decreased in CKD rats (P<0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). HNF4α binding to the CYP3A2 and CYP2C11 promoter regions was also decreased compared to controls (P<0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter and histone 3 acetylation in the CYP3A2 and CYP2C11 promoter locus for nuclear receptor activation. Conclusions: We demonstrate a novel mechanism of drug metabolizing enzyme regulation in CKD. Our results show that decreased CYP3A2 and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding and decreased histone acetylation in CKD. Adolescent Health and Educational Achievement: A Life Course Exploration of Twins and Siblings Elizabeth Wall-Wieler and Leslie L. Roos

Department of Introduction: Administrative data provide unique opportunities to examine the life course from different Community Health perspectives. Time-varying predictors, in conjunction with background variables, assess the development of several health and educational outcomes. For children between the ages of 14 and 18, important health Sciences, University of and education outcomes include major injuries, externalizing mental conditions, asthma, and high school Manitoba, Winnipeg, completion.. MB Objectives: To determine which background and time-varying covariates are important in predicting the outcomes, and in which age group the time-varying covariates are most significant. Methods: Multi-level modeling of administrative data from a cohort of twins and siblings born in Manitoba between 1984 and 1989 (n = 29,474) allowed comparing family and individual level characteristics controlling for unmeasured variables and using extensive sensitivity testing. Timevarying predictors include residential mobility, family structure, health measures, receipt of income assistance, receipt of services from Child and Family Services, and child taken into care by Child and Family Services. Background variables include: place of residence at birth (of oldest sibling), mother’s age at first birth, birth weight, gestational age and gender. Results: The predictors available provided the best fit for externalizing mental conditions (c-stat =0.9309) and high school graduation (c-statistic = 0.9192); the major injuries model (c-statistic = 0.7005) generated the poorest fit. The predictor with the greatest odds of a given health outcome between the ages of 14 and 18 was a diagnosis of that particular outcome in the previous age group (9-13). Family structure changes were most significant in reducing the odds of graduation when they occurred between the ages of 0 and 3; however, family structure changes during any age group significantly increased the odds of an externalizing mental conditions diagnosis between the ages of 14 and 18. Effects of the background variables varied with age and outcome studied. Conclusion: Childhood measures had significant, but varying, impact on health and educational outcomes in late adolescence. The importance of timing of exposure differed markedly among outcomes. Similarities between models predicting externalizing mental conditions and high school graduation highlight the need for a multidisciplinary orientation. Potent Inhibition of HIV-1 Infection by a Doxycycline-Inducible Lentiviral Vector Containing R88-APOBEC3GD128K Xiaoxia Wang, Zhujun Ao and Xiaojian Yao

Laboratory of Human APOBEC3G (A3G) protein is one of intrinsic anti-HIV restriction factors, which prevents viral Molecular Human replication by inducing detrimental mutant on intracellular proviral DNA and blocking viral reverse transcription. However, its antiviral action is counteracted by HIV-1 Vif, which blocks A3G encapsulation Retrovirology, with multiple mechanisms. To overcome Vif’s blockage, we previously generated an R88-A3G fusion Department of Medical protein, where A3G is efficiently delivered into Vif+ virus through R88. In this study, we further introduced Microbiology, Faculty the Vif-resistant mutant into fusion protein. Restriction by this optimized A3G: R88-A3GD128K, combined with suitable gene delivery system could provide a promising approach for anti-HIV-1 gene therapy. Here, of Medicine, University R88-A3GD128K was efficiently delivered into target cells with doxycycline (Dox) inducible lentiviral vector of Manitoba, Winnipeg, pTRIPZ (pTZ), where its expression and anti-viral activity were regulated by Dox. In the presence of Dox, the MB R88-A3GD128K modified T cells were resistant to infection of either laboratory or drug resistant virus isolates. + Importantly, induced R88-A3GD128K impaired viral replication in primary human CD4 T lymphocyte cells and peripheral blood mononuclear cells (PBMC) derived macrophages. Furthermore, to determine the action of R88-A3GD128K on chronically infection, we introduced R88-A3GD128K into ACH-2 cells. However, R88-A3GD128K did not disrupt phorbol 12- myristate 13- acetate (PMA) induced HIV-1 production. Infectivity of viruses produced from R88-A3GD128K transduced ACH-2 cells was significantly impaired, compared with viruses produced from control vector transduced cells. These data suggest that R88-A3GD128K is a highly potent HIV-1 inhibitor, and R88-A3GD128K based gene therapy is capable of blocking both active and latent infection. Variation in the Nicotine and Nitrosamine Metabolism Gene, CYP2A6, Alters Lung Cancer Risk among African American Smokers; Implications for Light Smoking and Minority Populations C.A. Wassenaar1, J. Knight2, M. Aldrich3, Q. Cai3, W.J. Blot3, R.F. Tyndale1,4,5

1Department of Introduction: We investigated the association of variation in the nicotine/nitrosamine metabolism gene, Pharmacology, CYP2A6, with the risk of developing lung cancer among African American cigarette smokers, a typically light smoking population at a high risk for lung cancer. Previously among European American smokers, we University of Toronto, showed that reduced/null activity CYP2A6 gene variants trended towards an association with lower lung 2Neuroscience cancer risk (OR 0.78; 95% CI 0.56-1.10) overall and significantly among those smoking less than 20 cigarettes Research Department, per day (CPD) (OR 0.63; 95% CI 0.41-0.97). Methods: We genotyped African Americans for reduced/null activity CYP2A6 alleles in a lung cancer case- CAMH, Toronto, ON, control study nested within the Southern Community Cohort, a prospective study investigating health 3Division of disparities. Controls were matched to incident lung cancer cases by age, gender, menopausal status and Epidemiology, recruitment site. We restricted this analysis to ever-smokers (n = 494; median CPD = 10; IQR 8-20) and grouped participants by CYP2A6 genotype into predicted reduced or normal metabolism groups (46% and Vanderbilt University, 54%, respectively). Lung cancer risk was estimated through conditional logistic regression modeling. Nashville, TN, Results: CYP2A6 reduced versus normal metabolizers had significantly lower lung cancer risk (crude OR 4Department of 0.61; 95% CI 0.40-0.93), and the association remained after adjusting for cigarette pack-years (OR 0.55; 95% CI 0.35-0.88). We found a significant interaction between CYP2A6 and pack-years (P = 0.05), which appeared Psychiatry, University to be driven by a greater difference in cancer risk by CYP2A6 genotype at lower pack-years. Median split of Toronto, Toronto, ON, analyses indicated that the CYP2A6-lung cancer association was significant among the lighter smokers: 5Campbell Family adjusted ORs 0.35, 95% CI 0.14-0.85 for ≤10 CPD and 0.63, 95% CI 0.25-1.55 for >10 CPD, but risk was not Mental Health higher among shorter than longer duration smokers. Conclusions: Our findings supportCYP2A6 as an important genetic risk factor among African American Research Institute, smokers and may be of relevance to other smoking populations with a high prevalence of reduced activity CAMH, Toronto, ON genotypes. Of note, genetic risk was prominent among light smokers, which is particularly relevant given the increasing prevalence of light and intermittent smoking in North America and the high rate of light smoking among many minority and aboriginal populations. Cancer Incidence in First Nations and Métis in Canada: Follow-up of the 1991 Census Mortality Cohort Diana Withrow

Dalla Lana School of Introduction: Cancer incidence amongst First Nations has historically been lower than that in the general Public Health, population, but preliminary province-level evidence suggests it is rising to meet or exceed that in the general population. University of Toronto, Objectives: We aim to estimate incidence rates of lung, prostate, breast and colorectal cancers in First Toronto, ON Nations and Métis (FN&M) and compare these to the rates in non-Aboriginal Canadians nationwide. Methods: A cohort of 1991 Long Form Census respondents was probabilistically linked to the Canadian Cancer Registry (1992 to 2003) and the Canadian Mortality Database (1992 to 2006). Age-standardized, site-specific incidence rates and time trends will be generated over the next 2 months and results will be available for presentation. This will be the first national study of cancer incidence in FN&M. Further, this linked cohort provides a rare opportunity to include groups typically under-represented in health research (e.g. Métis and non-Status Indians) since FN&M will be identified based on self-report of Aboriginal ancestry, Registration and band membership in the Census. Results: The cohort includes 2.7 million adults 25 and older on Census day in 1991, equivalent to a 15% sample of the population of that age at the time. Of these, approximately 62,000 reported either First Nations ancestry or are registered under the Indian Act of Canada. Preliminary analyses show that crudely, cancer incidence from 1992 to 2003 was higher in non-Aboriginals than in FN&M nationwide. FN&M had a similar incidence of breast and colorectal cancers, both lower than the general population. The Métis have significantly lower incidence of prostate cancer than the general population, but significantly higher rates than First Nations. Incidence rates of lung cancer in Métis are comparable to those in the general population, while First Nations have lower lung cancer incidence. Conclusions: Using an innovative approach, this study will provide much needed data to inform effective, culturally-appropriate cancer prevention and control activities for FN&M in Canada. New cancer incidence data will help justify and re-prioritize FN&M cancer programs by identifying issues that require the most attention. Superantigen-Mediated Immune Suppression in the Liver during Staphylococcus aureus sepsis Stacey X. Xu1, Miren L. Baroja1,2, Kelly Summers1,2, S.M. Mansour Haeryfar1, and John K. McCormick1,2

1Department of Staphylococcal superantigens (SAgs) are potent immunomodulatory toxins capable of activating massive Microbiology and amounts of T cells resulting in the over-release of cytokines, which can lead to the development of toxic shock syndrome. While the structure and function of SAgs have been elucidated in great detail, the Immunology, purported biological role of these toxins has never been completely explained. A SAg knockout was University of Western created in S. aureus to explore the role of SAgs during sepsis. Mice infected with wild type S. aureus had Ontario, London, ON, statistically significant higher bacterial loads in the liver and heart, and increased weight loss, compared to mice infected with SAg-deficient S. aureus. Flow cytometry analysis showed that mice infected with 2London Health wild type S. aureus had decreased Vβ3 T cell populations in the lymph nodes, demonstrating that the SAg Research Institute, staphylococcal enterotoxin A (SEA) was produced in vivo which has important clinical implications. In London, ON contrast, mice infected with SAg-deficient S. aureus had Vβ3 levels comparable to uninfected controls. This data suggests that SAg-deficient S. aureus are more likely to be cleared during infection than their SAg- producing counterparts. Flow cytometry indicates that there is a higher percentage of F4/80+, CD11c+, and CD3+ leukocytes in SAg-deficient infected mouse livers, but a lower percentage of Gr-1+ and CD11b+ cells than leukocytes from SAg-producing infected mouse livers. Multiplex analysis also showed higher levels of IFN-g and MCP-1 in WT-infected livers. Investigations are underway to elucidate this mechanism further and the cell subtypes involved. The results of this study may lead to the recognition of a novel mechanism of immune suppression by SAgs. This is significant as most studies in the field have concentrated mainly on T cell anergy, whose physiological role is yet to be proven clinically. By unmasking a potential new mechanism of pathogenesis, we can develop better therapies to combat staphylococcal infections. CLA Isomers Modify Adipokine Secretion during Adipogenesis Azadeh Yeganeh1,3 Carla G. Taylor1,2,3 Jenna Poole 3 Peter Zahradka 1,2,3

Departments of Background: Obesity is a major health problem worldwide, and it is associated with other diseases such 1Physiology and as insulin resistance, dyslipidemia, and hypertension. There are many natural products that have the ability to control the processes associated with obesity, and conjugated linoleic acid (CLA) has been reported to 2 Human Nutritional have positive effects. Numerous studies have demonstrated that t10,c12-CLA treatment during adipocyte Sciences, University of differentiation reduces lipid accumulation in both mouse and human adipocytes, although the mechanism Manitoba, Winnipeg, of action is unknown. The Wnt signalling pathway has a major role in both commitment and differentiation of adipocytes. Specifically, Wnt stimulates the commitment of stem cells to the adipocyte lineage (pre- MB, 3Canadian Centre adipocyte); however, it inhibits the differentiation of pre-adipocytes to mature adipocytes. for Agri-food Research Objectives: This study examined the effect of CLA isomers on adipokine secretion at different time points in Health and during differentiation. We also investigated the role of CLA isomers during the early stages of adipogenesis. In addition, the potential role of Wnt/ -catenin signaling in the anti-adipogenenic action of CLA was Medicine, St. Boniface β examined. Hospital Research Methods: 3T3-L1 cells were used as an model of adipocyte differentiation. Adipogenesis was induced by Centre, Winnipeg, MB treating 3T3-L1 preadipocytes with a cocktail of insulin, dexamethasone and methylisobuthylxanthine; the c9,t11 and/or t10,c12-CLA isomers (60 μM) were added concurrently with the adipogenic cocktail. Cells were lysed at 0, 2, 4 or 8 days after differentiation. The medium was changed every 2 days and the treatments were refreshed at the same time. Western blotting was employed to measure the expression of target proteins. Result: We have shown that both c9-t11 and t10-c12 CLA isomers stimulate pre-adipocyte differentiation at an early stage. We also confirmed that CLA isomers affect adipokine secretion from mid-differentiation onwards. The two isomers do not have the same effect: c9-t11 CLA increases adipokine secretion while t10-c12 CLA inhibits adipokine secretion. We also observed that t10-c12 CLA stimulates phosphorylation of LRP6/5 (co-receptor of Wnt signaling) in 3T3-L1 cells. Conclusion: Our data suggest that t10,c12-CLA activates Wnt-β-catenin signaling in 3T3-L1 adipocytes. Novel MeCP2E1 Antibody Reveals Neural Cell Type Specific Expression Robby Zachariah, Carl Olson, Chinelo Ezeonwuka, and Mojgan Rastegar

Regenerative Medicine Methyl CpG Binding Protein 2 (MECP2) is an epigenetic regulator capable of recognizing and binding to Program, Department methylated DNA. Mutations in MECP2 are most frequently associated with Rett Syndrome patients. Rett Syndrome is a progressive neurological disease affecting young females and occurs with an incidence of of Biochemistry and 1 in 10,000 live births. Alternative splicing of MECP2 leads to the formation of two isoforms, MECP2E1 and Medical Genetics, MECP2E2. Mutations in gene regions exclusively coding for MECP2E1 isoform has been reported to be Faculty of Medicine, sufficient to cause Rett Syndrome. Furthermore, MeCP2E1 has also been shown to rescue RTT phenotypes in mouse models. However, studies on MeCP2 isoforms have been limited due to the unavailability of University of Manitoba, commercial antibodies that differentiate between the two isoforms. Although MeCP2 is known to be Winnipeg, MB expressed in all cell types constituting the brain, recent studies have shown that many RTT phenotypes are linked specifically to neurons. In the current study, we have generated for the first time, an antibody that specifically detects MeCP2E1. We have also demonstrated the applicability of our antibody in various techniques including immunocytochemistry, immunohistochemistry and western blotting. Using our custom MeCP2E1 antibody, we have elucidated the neural cell-type specific expression pattern of MeCP2E1. Our results provide novel insights into the etiology of Rett Syndrome and the molecular functions of MeCP2 isoforms. Gold Nanoparticle in Targeted Chemotherapy for Melanoma Xuan Zhang1, Hicham Chibli1, Jose Teodoro2, Jay Nadeau1

1Department of Being widely applied in clinical cancer treatments, chemotherapy still remains limited in melanoma Biomedical treatment due to this cancer’s strong resistance. The most well known resistance mechanisms include melanosomal quenching, drug efflux, and an enhanced self-repair system. As a result, this inherited Engineering, McGill resistance has significantly lifted the effective dosage of therapeutics, thus raising the risk of side effects University, such as severe cardiotoxicity. Therefore, modifications on existing chemotherapeutics to overcome this 2Department of problem will not only help generalize a method to upgrade drugs in application, but also help reduce the cost for new drug research and development. Biochemistry, McGill Gold nanoparticles have the intrinsic advantages in enhancing the efficacy of chemotherapeutics such University, Montreal, QC as Doxorubicin for targeted treatment. They are chemically inert with high solubility, and the tunable size enables them to be involved in Enhanced Permeability and Retention (EPR) effect for passive targeting. The various surface groups also render the particles to be functionalized with cancer specific molecules for active targeting. In this work, we developed a drug complex (AuDox) involving gold nanoparticles and doxorubicin (Dox) to address the resistance problem. Ultrasmall gold nanoparticles (2.8nm) were synthesized, and conjugated to Dox via a stable amide bond at a ratio of 1:25. Surprisingly, the IC 50 value of Dox dropped by almost 20 fold while the gold particles remain nontoxic. Phenotypic analyses showed that the AuDox penetrated the cell membrane and concentrated in the nuclei at a much faster rate, and that they could also escape the constant exportation by the ABC-transporter involved in multidrug resistance. Both apoptosis and necrosis were observed to be associated with AuDox cytotoxicity mechanisms. New in-vivo results indicated that the AuDox complex was able to reduce the tumor growth rate of the aggressive B16 melanoma at 1/10 of the Dox effective dosage. The current focus is on a genome-wide loss-of-function screen. It will help us unveil how the conjugates overcome this resistance on a genotypic level. ALS-Associated FUS Mutations Result In Compromised FUS Alternative Splicing And Autoregulation Yueqin Zhou, Songyan Liu, Arzu Ozturk, Geoffrey G. Hicks

Manitoba Institute of Introduction: The RNA binding protein FUS/TLS is the third most frequently mutated gene in amyotrophic Cell Biology, lateral sclerosis (ALS). Mutations commonly result in varying deficiencies of FUS nuclear localization, which predicts a compromised function in RNA processing. This study is the first to demonstrate alteration of FUS- Department of regulated RNA processing for biologically relevant targets and its contribution to ALS pathogenesis. Biochemistry and Methods: Crosslinking immunoprecipitation with deep sequencing (CLIP-seq) and bioinformatics analysis Medical Genetics, was used to identify RNA targets of FUS. Analysis of splicing variants by PCR was performed for both the splicing reporter minigene and the endogenous transcript. siRNA knockdown, overexpression and University of Manitoba, mutagenesis of FUS was used for functional tests. Western blot was used to analyze protein levels. Winnipeg, MB Results: We identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNA using CLIP-seq. Our data demonstrated that FUS is a repressor of exon 7 inclusion and that the splice variant with exon 7 skipped is subject to nonsense-mediated decay. Over- expression of FUS leads to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, knockdown of endogenous FUS protein increases exon 7 inclusion. The data suggest an autoregulatory mechanism which controls FUS homeostasis by a feedback loop. We next demonstrated that the increasing nuclear localization deficiency of ALS-associated FUS mutants R521G, R522G orΔ Exon15 (minor, moderate or severe, respectively) correlates with a respectively increasing compromise of both exon 7 repression and FUS autoregulation. Compromised FUS autoregulation results in a feed-forward loop to exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. Moreover, we showed that antisense oligonucleotides can be used to induce FUS exon 7 skipping, indicating the potential to alleviate abnormal FUS cytoplasmic accumulation in ALS. Conclusion: Here we report a novel FUS autoregulatory mechanism by alternative splicing and its deficiency in ALS-associated mutants. This data is the first to demonstrate mechanistically that the alteration of a FUS-regulated RNA processing event directly contributes to ALS pathogenesis. It suggests the altered RNA processing of a significant number of genes regulated by FUS is a major mechanism underlying ALS pathogenesis. APP/PS1 Mutations Sensitize Oligodendrocytes to Amyloid Toxicities, and Exacerbate White Matter Damage and Memory Impairment in Mice Shenghua Zhu1, Junhui Wang2, Jinping Qiao2, Xin-Min Li1,2

1Department of Introduction: Mutations in the genes encoding amyloid precursor protein (APP) and presenilin 1 (PS1) Pharmacology and cause some cases of early-onset inherited AD which is so called familiar AD. These mutations may promote neuronal degeneration by increasing the production of neurotoxic forms of amyloid β-peptide (Aβ) and by Therapeutics, perturbing synaptic integrity. Oligodendrocytes play a critical role in brain function by myelinating axons 2Department of and providing trophic support to the axons. Damage to white matter occurs in the brains of patients with Psychiatry, University AD, but it is not known if and how oligodendrocytes are affected in AD, nor whether white matter alterations contribute to the cognitive dysfunction in this disease. of Manitoba, Winnipeg, Methods: Eight-week-old APPK670N/M671L/PS1M146L mutant knockin mice and littermate wild-type (WT) MB mice were fed either normal mouse chow or chow containing 0.2% (w/w) cuprizone (CPZ) by weight for 5 weeks to induce demyelination. The demyelinated mice were allowed to recover for 0, 2, 3, 4 or 6 weeks. We performed behavioural tests, immunohistochemistry, western blot, Elisa and electron microscopy at each time point to analyze the memory and the expression of myelination and Aβ in the animal model. Results: 5 weeks damage to oligodendrocytes induced by cuprizone is not significantly different between APP/PS1 mutant knockin mice and wild-type controls, nor was any behavioural difference observed. Nevertheless, after 6 weeks of spontaneous remyelination cuprizone treated APP/PS1 knockin mice not untreated APP/PS1 mice showed early symptoms of cognitive function decline, in particular the hippocampus related long term memory, without exhibiting any obvious change of the amyloid plaques. More interestingly, toxic oligometric Aβs in both the brain and serum were significantly increased in the cuprizone treated APP/PS1 knockin mice, indicating the permeability of blood brain barrier was to some extend compromised due to the damaged oligodentrocytes. Furthermore, during the 6 weeks recovery period, APP/PS1 knockin mice exhibited a significantly slower rate of the repopulation of mature oligodendrocytes in the demyelinated lesions, which was associated with downregulation of transcription factor olig2 in the process of cell maturation. Conclusion: These findings suggest a possible mechanism responsible for white matter damage in AD and a contribution of such damage to cognitive impairment. 2013 sponsors AFMC Faculties of Medicine: McGill University Memorial University of Newfoundland Queen’s University University of Alberta University of British Columbia University of Calgary Université de Laval University of Manitoba University of Ottawa University of Saskatchewan University of Toronto Western University Apotex Fermentation Inc. Children’s Hospital Foundation Inc. Canadian Institutes of Health Research (CIHR) CIHR Institute of Cancer Research CIHR Institute of Genetics CIHR Institute of Infection and Immunity Office of the President Dean of Graduate Studies, University of Manitoba Dean of Medicine, University of Manitoba E.L. Drewry Memorial Trust Health Sciences Centre Foundation, Inc. Heart and Stroke Foundation of Manitoba Manitoba Health Research Council Manitoba Institute for Child Health Manitoba Medical Service Foundation Inc. Manitoba Innovation, Energy and Mines, Government of Manitoba Physician Resource Coordination Office (PRCO), Government of Manitoba St. Boniface General Hospital Foundation, Inc. S.W. Prowse Memorial Trust Vice-President (Research), The University of Manitoba

Faculty of Medicine